In vivo whole cell mathematical model for
metabolic reaction network for model
organism Penicillium chrysogenum
Raymond Blankestijn
MSc. Thesis
Biochemical Engineering
Delft University of Technology
Faculty of Applied Sciences
Department of Biotechnology
BioProcess Technology (BPT) group
Supervisors:
I.E. Nikerel MSc.
Dr. ir. P.J.T. Verheijen
Dr. ir. W.A. van Winden
Dr. W.M. van Gulik
Prof. dr. ir. J.J. Heijnen
Abstract1
Biological systems present a complexity beyond intuitive comprehension and to obtain a better
understanding of the behaviour of the living organisms, large scale dynamic mathematical models are
employed. From a system biology perspective, these models should not only describe the kinetic behavior
of metabolic reaction networks that feature metabolite-enzyme interactions (allosteric feedback or feed
forward), intercompartmental transport, and cofactor coupling, but, they should also ultimately allow
combining several pathways (horizontal modeling) and/or “omic” levels (vertical modeling) in the cell.
However, currently by far most of the available models are limited to only one pathway and one “omic”
level.
In this work, a large scale kinetic metabolic model of Penicillium chrysogenum is developed, which aims
to encompassing all the major pathways present in the organism. In constructing the kinetic model, we
first define the stoichiometric network presented in van Gulik et al. (2000), which consists of 188
metabolites and 167 reactions located in 3 compartments (cytosol, mitochondria and peroxisome) and
postulated a kinetic expression for each of the reactions. We used approximative linlog kinetics for the
rate expressions, which allowed us to represent the enzyme-metabolite kinetic interactions by an elasticity
matrix. Information on the presence and absence of mass action and allosteric enzyme kinetic information
was obtained from literature survey and database search. The final values of the elasticities needed to be
estimated by fitting the model to the available short term kinetic response data.
Two major limitations have been encountered regarding the measurements of metabolites: (1)
measurement of a metabolite at all and (2) compartmentation, i.e. measurements of metabolites/reactions
that are present in multiple compartments (Nasution et al. 2006). To deal with the compartmentation
problem, the system has been reduced 89 metabolites and 75 reactions within one compartment, by
lumping, using insights gained both from biochemical knowledge and from data recently published by our
group on short term kinetic responses of primary metabolism of Penicillium chrysogenum (Nasution et al.
2006). The limited number of available measurements was dealt by data-driven model reduction while
applying the parameter estimation scheme to the large model. To estimate the kinetic parameters, the aim
1
This work has been accepted for the European Congress of Chemical Engineering -6, Copenehagen 16-21
September
ii
was to followed the methodology presented in Nikerel et al. (2006) in which the theory was applied to a
small example system. However due to time constrains no kinetic parameter has been estimated.
The cover shows a screenshot of the penG model implemented in the software program In Silico
iii
Table of Contents
Abstract ........................................................................................................................................... ii
Table of Contents ............................................................................................................................iv
1
Introduction ..............................................................................................................................1
1.1
Why use mathematical modelling ....................................................................................1
1.2
Challenges in mathematical modelling ............................................................................2
1.3
Background of the model organism Penicillium chrysogenum .......................................4
1.4
Aim of project ..................................................................................................................6
2
Methods....................................................................................................................................7
2.1
Stoichiometric model Penicillium chrysogenum .............................................................7
2.2
Background stoichiometric matrix and steady state rates ................................................8
2.3
The linlog format..............................................................................................................9
2.4
Enzyme-metabolite connectivity....................................................................................10
2.5
Model Simplification......................................................................................................12
2.6
Model Reduction ............................................................................................................13
2.6.1
Data-driven reduction of metabolic network .........................................................13
2.6.2
Identification of PEQ reactions ..............................................................................14
2.6.3
Calculation of unmeasured metabolites from PEQ rates. ......................................16
2.6.4
Time scale analysis.................................................................................................23
2.6.5
Pseudo Equilibrium (PEQ).....................................................................................24
2.6.6
Pseudo Steady State (PSS) .....................................................................................28
Experimental Part...........................................................................................................................33
2.7
Computational tools .......................................................................................................33
3
Results ....................................................................................................................................34
3.1
Calculation of net conversion rates ................................................................................34
3.1.1
Balancing net conversion rates...............................................................................35
3.1.2
Adjusting biomass definition .................................................................................38
3.2
Model Reductions ..........................................................................................................40
3.2.1
Data driven reduction of stoichiometric model......................................................40
3.2.2
Lumping and adjusting the oxidative phosphorylation ..........................................48
3.2.3
Calculation of unmeasured metabolites .................................................................50
3.2.4
Checking PEQ assumptions ...................................................................................51
3.2.5
Elimination of PEQ rates and new defined equilibrium pools...............................54
3.2.6
Turnover times .......................................................................................................56
3.2.7
PSS metabolites......................................................................................................59
3.2.8
Frozen Metabolites.................................................................................................61
3.3
Postulation of Reaction Kinetics ....................................................................................61
3.3.1
Glycolysis...............................................................................................................61
3.3.2
Pentose phosphate pathway....................................................................................62
3.3.3
TCA cycle ..............................................................................................................63
3.3.4
Anaplerotic pathways.............................................................................................64
3.3.5
Oxidative phosphorylation .....................................................................................64
3.3.6
Transfer of 1-C compounds....................................................................................65
3.3.7
Transport across plasma membrane .......................................................................65
iv
3.3.8
Amino acid synthesis .............................................................................................65
3.3.9
Nucleotide biosynthesis..........................................................................................71
3.3.10
ATP hydrolysis.......................................................................................................72
3.3.11
Synthesis of glycogen and polysaccharides ...........................................................72
3.3.12
Biomass formation .................................................................................................73
3.3.13
Penicillin biosynthesis............................................................................................73
3.3.14
Summary ................................................................................................................73
4
Discussion ..............................................................................................................................75
5
Conclusion..............................................................................................................................78
6
Nomenclature .........................................................................................................................79
7
References ..............................................................................................................................81
Appendix A: Net Conversion Rates ............................................................................................ - 1 Appendix B: Lumped Reactions ................................................................................................. - 5 Appendix C: Connectivity metabolic network.......................................................................... - 15 8
Lumped metabolites .......................................................................................................... - 15 9
Glycolysis.......................................................................................................................... - 16 10
Pentose phosphate pathway........................................................................................... - 21 11
TCA cycle ..................................................................................................................... - 24 12
Anaplerotic pathways.................................................................................................... - 30 13
Oxidative Phosphorization ............................................................................................ - 31 14
Different carbon substrates............................................................................................ - 32 15
Transfer of 1-C compounds........................................................................................... - 32 16
Transport across the plasma membrane ........................................................................ - 34 17
Transport across the mitochondrial membrane ............................................................. - 34 18
Amino acid synthesis .................................................................................................... - 34 18.1 Glutamate pathway.................................................................................................... - 34 18.2 Proline biosynthesis................................................................................................... - 35 18.3 Arginine biosynthesis................................................................................................ - 36 18.4 Lysine biosynthesis ................................................................................................... - 39 18.5 Serine biosynthesis.................................................................................................... - 42 18.6 Glycine biosynthesis ................................................................................................. - 43 18.7 Sulfur metabolism ..................................................................................................... - 43 18.8 Alanine & aspartate metabolism ............................................................................... - 47 18.9 Threonine biosynthesis.............................................................................................. - 48 18.10
Methionine biosynthesis........................................................................................ - 50 18.11
Leucine, isoleucine & valine metabolism ............................................................. - 51 18.12
Phenylalanine, tyrosine and tryptophan biosynthesis ........................................... - 56 18.13
PRPP biosynthesis................................................................................................. - 62 18.14
Histidine biosynthesis ........................................................................................... - 62 19
Protein synthesis............................................................................................................ - 64 20
Nucleotide biosynthesis................................................................................................. - 64 20.1 Purine metabolism (IMP synthesis) .......................................................................... - 64 20.2 Pyrimidine metabolism ............................................................................................. - 69 21
RNA synthesis............................................................................................................... - 72 22
ATP hydrolysis.............................................................................................................. - 72 23
Synthesis of fatty acids.................................................................................................. - 73 23.1 Inositol phosphate metabolism.................................................................................. - 73 v
23.2 Glycerophospolipid metabolism ............................................................................... - 74 23.3 Glycerolipid metabolism ........................................................................................... - 77 23.4 Miscellanous.............................................................................................................. - 78 23.5 Biosynthesis of steriods............................................................................................. - 79 24
Synthesis of glycogen and polysaccharides .................................................................. - 83 24.1 Aminosugar metabolism ........................................................................................... - 83 24.2 Fructose and mannose metabolism ........................................................................... - 85 24.3 Starch and sucrose metabolism ................................................................................. - 86 24.4 Miscellanous.............................................................................................................. - 87 25
Biomass formation ........................................................................................................ - 87 26
Penicillin biosynthesis................................................................................................... - 88 27
Transport across the peroxisomal membrane................................................................ - 90 28
References ..................................................................................................................... - 91 Appendix D: Pool composition matrix...................................................................................... - 92 Appendix E: Transient behaviour unmeasured metabolites...................................................... - 93 Appendix F: Matlab files & In Silico database (part of)........................................................... - 95 -
vi
1 Introduction
In this chapter an introduction is given which discusses the usefulness of developing a mathematical
model for a complex metabolic network and the potential difficulties and challenges associated with it.
The model organism in this study is Penicillium chrysogenum which is discussed in chapter 1.3.
1.1 Why use mathematical modelling
Mathematical modelling complex metabolic reaction networks and its regulation is indispensable to
achieve better insight of the behaviour of a living cell. It allows the quantitative examination of the
system, so that the metabolic network characteristics can be predicted, designed or optimized. This
information can be used for experimental design or metabolic engineering in order to optimize the
functioning of the model organism in an industrial process.
Before a mathematical model is developed, the system boundaries must be specified, which separates
important components from their environment. Defining the system boundaries enables the distinction
between outside the model, the so-called exogenous variables, and inside the model, the so-called
endogenous variables. The endogenous variables need to be specified, in order to model the exogenous
variables. In this project the focus lies on the metabolite-enzyme interactions and the dynamics of the
metabolites and as a consequence the system is restricted by the metabolome level and neglects the
interaction between the genome, transcriptome and proteome level. Hence it is a simplification of the
reality as it does not include changes in gene expression or enzyme levels.
The mathematically model of a metabolic reaction network can be divided by the following main model
classes:
•
static models
•
dynamic models
A static model describes the system with time independent data using steady state values and enables the
examination of structural characteristics, e.g. flux distributions and limiting fluxes, with tools like
metabolic flux analysis and metabolic network analysis. For static models the dx/dt and dc/dt are set to
zero, see equation (1.1) & (1.2). A dynamic model describes the system with time dependent data and
should be able to predict the dynamic behaviour of the components.
Briefly, in order to develop a mathematically model for a metabolic network, it is necessary to
•
define the stoichiometry of all reactions, expressed in the stoichiometric matrix S,
•
postulate the reaction kinetics,
1
•
set up the proper mass balances for each metabolite.
The mass balance of the involved metabolites in the metabolic network, which can be written as a set of
differential relations:
dx
= Sx ⋅ v
dt
dc
= S c ⋅ v + ( Din ⋅ c feed − Dout ⋅ c )
dt
(1.1)
(1.2)
Sx denotes the stoichiometric matrix containing intracellular metabolites, Sc denoted the stoichiometric
matrix containing extracellular metabolites, v is a column vector of the metabolic fluxes, cfeed a column
vector of the influent concentration of extracellulars, c the column vector of the concentration of
extracellulars in the reactor, x the column vector of the concentrations of intracellulars and Din & Dout are
the dilution rates of the influent and effluent. These differential equations describe the time dependent data
obtained from biological system.
The dilution rate can be calculated by dividing the volumetric flow rate, written as F, with the volume of
the system considered, written as V.
D=
F
V
(1.3)
A mathematical metabolic model contains many variables and parameters, which requires a numerical
calculation as an analytical solution may not be obtained.
In the next chapter the challenges in developing a mathematical model are discussed.
1.2 Challenges in mathematical modelling
The challenge in constructing a mathematical metabolic model is postulating the reaction kinetics for the
reactions and acquiring reliable rate kinetic parameters which can be used for in vivo conditions.
The reaction rate, written as v, is commonly assumed to be proportional to the enzyme level, e, and a nonlinear function of the metabolite concentrations, x & c, and enzyme kinetic parameter p.
v = f ( e, x , c, p )
(1.4)
Several reaction rate formats have been derived which can be distinguished into:
•
Mechanistic rate kinetics
•
Approximative rate kinetics
Examples of mechanistic kinetics are mass action, Michealis-Menten and Hill kinetics. Examples of
approximate kinetic formats are GMA type power law and linlog, see Table 1.1 (Heijnen 2005).
2
Table 1.1: Reaction kinetics formats
Mass action:
Michaelis-Menten:
Hill:
(1.5)
v i =k cat X 1 X 2
vi =
Vmax [ E ][ X 1 ]
[ X1 ] + KM
(1.6)
Vmax [ X 1 ]
(1.7)
h
vi =
[ X 1 ]h + K h
Linear in metabolite and enzyme level:
v
⎛e ⎞
⎛ x ⎞
⎛c ⎞
- i = ⎜ 0 - 1 ⎟ + E0x ⋅ ⎜ 0 - i ⎟ + E0c ⋅ ⎜ 0 - i ⎟
0
J
⎝e
⎠
⎝x
⎠
⎝c
⎠
(1.8)
Log-linear in metabolite and enzyme level:
v
⎛e⎞
⎛ x⎞
⎛c⎞
- i = ln ⎜ 0 ⎟ + E0x ⋅ ⎜ 0 ⎟ + E0c ⋅ ⎜ 0 ⎟
0
J
⎝e ⎠
⎝x ⎠
⎝c ⎠
(1.9)
Linear in metabolite levels
v ⎛e⎞ ⎛
⎛ x
⎞
⎛c
⎞⎞
= ⎜ 0 ⎟ ⋅ ⎜ 1 + E0x ⋅ ⎜ 0 − 1⎟ + E0c ⋅ ⎜ 0 − 1⎟ ⎟
0
J
⎝e ⎠ ⎝
⎝x
⎠
⎝c
⎠⎠
(1.10)
GMA type power law
⎛ v⎞
⎛e⎞
⎛ x⎞
⎛c⎞
ln ⎜ 0 ⎟ = ln ⎜ 0 ⎟ + E0x ⋅ ⎜ 0 ⎟ + E0c ⋅ ⎜ 0 ⎟
⎝J ⎠
⎝e ⎠
⎝x ⎠
⎝c ⎠
(1.11)
Linlog
see chapter 2.3
Abbreviations: vi = reaction rate of reaction i, X1 and X2 metabolite concentration of species 1 and 2 respectively, kcat = reaction rate constant, E
enzyme level, Km Michaelis-Menten constant, Vmax = maximum velocity, h = Hill coefficient, K = Hill constant, J0 = column vector steady state
metabolic fluxes, e column vector enzyme levels, Ex0 = elasticity matrix of intracellular metabolites, Ec0 = elasticity matrix of extracellular
metabolites, and i = identity matrix. The superscript 0 denotes the reference state, i.e. the steady state value.
Traditionally the kinetics of these enzymatic reactions is commonly based on data obtained from in vitro
kinetic studies, which are based on test tube tests of purified enzymes and the obtained kinetics are nonlinear with a lot of parameters. However translation to in vivo condition, i.e. the physiological conditions
of the cell, which includes phenomena such as buffering conditions, homeostatis, molecular crowding, is
difficult because there are several number of differences between the in vitro and the in vivo conditions,
such as:
•
many other metabolites are present in in vivo conditions, which might influence the enzyme
activity,
•
the in vivo enzyme levels are significantly higher, which might cause enzyme-enzyme
interactions,
•
the substrate to enzyme ratio of in vivo lies in a completely different range than under in vitro
conditions, caused by the low in vivo substrate concentrations.
3
It has been shown that applying in vitro determined kinetic parameters does not result in the correct
prediction of the dynamic in vivo behaviour and requires the estimation of kinetic parameters based on in
vivo data (Teusink et al. 2000). The identification of kinetic parameters can be done by using transient in
vivo metabolite measurements. An example of this process is given by Rizzi et al., who developed a model
to estimate the parameters in their mathematical model that describes the glycolysis and TCA cycle of
Saccharomyces cerevisiae (Rizzi et al. 1997). In their approach, mechanistic rate equations are employed
which results that the kinetic model is complicated due to its non-linearity and significant number of
parameters.
The kinetic model can be simplified by using approximative rate equations which is linear in its kinetic
parameters, i.e. the elasticities. The elasticity matrixes Ex0 & Ec0 shown in Table 1.1, contain the
elasticities coefficients of intracellular and extracellular metabolites respectively. The enzyme-metabolite
interaction is represented by the elasticity coefficient, which is defined as the change in reaction rate
caused by an infinitesimal change in the involved effectors, inhibitors and activators, normalized to a
reference condition:
x 0j ⎛ dvi ⎞
ε = 0 ⋅⎜
⎟
vi ⎜⎝ dx j ⎟⎠
0
0
ij
(1.12)
The elasticity is negative for inhibitors, positive for activators and zero in case no enzyme-metabolite
interaction exists and can be estimated from the transient in vivo metabolic data obtained from a dynamic
perturbation experiment, as described in Nikerel et al. (2006).
However also the size and structure of the metabolic reaction network can cause problems, since the
amount of data is often limited, i.e. only for a few metabolites the transient behaviour has been measured.
In a model-driven approach, the kinetic parameters of the metabolic model are identified with ‘brute
force’ in which m kinectic parameters are estimated with information available on n parameters and for
large systems n is frequently significant smaller than m. In a data-drive approach, the metabolic model is
reduced by lumping the unmeasured and unimportant metabolites after which the kinetic parameters are
estimated. Many of these metabolites can not be measured due to their concentrations in the cell.
Hence the challenge is to develop a mathematical model which requires a minimum of kinetic parameters
and should describe transient measurements.
1.3 Background of the model organism Penicillium chrysogenum
In this project the model organism Penicillium chrysogenum is selected since it has been intensively
studied in our laboratory. The organism serves as the source of the antibiotic penicillin which eliminates
4
gram-positive bacteria. It disrupts the cell wall synthesis by inhibition of the transpeptidaase enzyme
which prevents the cross linking of the peptidoglycan polymers. The resulting malformed cell walls take
on excess water, which causes them to burst, i.e. cell lysis. The organism thanks its name to the similarity
of the conidiophore of the fungus to a paintbrush, which in Latin is called penicillus.
Figure 1.1: Strain of Penicillium chrysogenum
Alexander Fleming discovered penicillin by accident in 1928 after he discovered a blue-green mould
covering his Staphyloccoccus bacterial specimens in a petri dish. After examining the mould under a
microscope, he noticed that the bacteria surrounding the mould were dead or dying due to the fact that the
mould prevented the bacteria to make and regenerate new cell walls. (Diggins 1999).The mould was
identified as Penicillium notatum which was later redirected to Penicillium chrysogenum.
Penicillium chrysogenum is the main source for the industrial and clinical important β-lactam antibiotics,
such as penicillin G, ampicillin, amoxicillin and cephalexin, which are used to treat bacterial infections.
Today, β-lactam antibiotics are one of the major biotechnology products with a turnover of approximate
11.4 billion euros (Elander 2003) and due to the hard competition from low labour costs countries in the
Far East, it becomes more and more important to reduce the energy and material costs of its production
which can partly be achieved by improving the biosynthesis of penicillin by Penicillium chrysogenum.
Developing a dynamic mathematical metabolic model is important to identify potential bottlenecks for the
biosynthesis of penicillin and provides explanatory simulations to enhance the understanding of the living
cell. This information can be employed for metabolic engineering, which aims the optimization of genetic
5
and regulatory processes in order to enhance the biosynthesis of a certain component of interest such as
penicillin.
1.4 Aim of project
The kinetic model is based on the stoichiometric reaction network that is developed in (van Gulik et al.
2000), which includes major pathways such as the glycolysis, pentose phosphate pathway (PPP),
tricarboxylic acid cycle (TCA) and secondary pathways such as the amino acid and nucleotides
biosynthesis. The metabolic network contains 188 metabolites and 167 reactions located in 3
compartments, i.e. cytosol, mitochondria and peroxisome, see appendix B.
In developing a model, two major limitations on transient metabolite measurements are encounters:
•
limited number of available measurements
•
compartmentation, measurements the average concentration of the whole cell and of the
individual concentrations in the compartments.
These limitations have been dealt with by a data-driven model reduction of the metabolic reaction
network. Furthermore reactions that can be assumed to be near equilibrium, allow the calculation of some
unknown metabolites.
Approximative linlog kinetics has been used as the kinetic format which allows the metabolite-enzyme
interactions to be expressed in an elasticity matrix. Information about allosteric and feed back inhibition
has been obtained from a literature survey and a database search (Brenda 2006; KEGG 2006; Metacyc
2006; SabioRK 2006), which can be found in appendix C. A priori information about the enzymemetabolite interactions can be used to assign non-zero elasticities which can be estimated by time
dependent perturbation data, presented in Nasution (2007). The main results are summarized in appendix
A. Time scale analysis is employed to further reduce the model, which is discussed in chapter 2.6.
The aim of the project is to develop a dynamic mathematical model for the model organism Penicillium
chrysogenum using approximated linlog kinetics. The model is based on the metabolic reaction network
published in van Gulik et al. (2000) and should describe the time dependent perturbation data obtained
from Nasution et al. (2006) so that the network characteristics can be determined and the kinetic
parameters can be estimated according to Nikerel et al. (2006).
6
2 Methods
The construction of a kinetic metabolic model consists of several steps which are schematically shown in
Figure 2.1. The first step is to obtain a stoichiometric model that briefly will be discussed in chapter 2.1.
Subsequently the stoichiometric matrix can be constructed and the steady state metabolic fluxes can be
calculated which is discussed in chapter 2.2.
Once the metabolic reaction network has been fully defined, the reaction kinetics can be postulated using
the linlog kinetic format. This will be based on the findings of the enzyme-metabolite interactions from
literature studies, online database searches and personal communication, see chapter 2.4.
The next step is to reduce the model which includes a data-drive reduction of the metabolic network,
identification of reactions that are close to equilibrium and time scale analysis. This will be discussed in
chapter 2.6.
Definition of
stoichiometric
model
Postulation of
kinetic effect
Model Reduction
Parameter
estimation
Simulation
Figure 2.1: Schematic overview of different steps involving the development a large kinetic model
Subsequently the kinectic parameters can be estimated (Nikerel et al. 2006) after which the system can be
simulated.
2.1 Stoichiometric model Penicillium chrysogenum
van Gulik et al. developed a stoichiometric model for growth and penG production in Penicillium
chrysogenum (van Gulik et al. 2000). The stoichiometric model for the growth on glucose contains 188
metabolites and 167 reactions located in 3 compartments. The stoichiometric model describes the central
metabolism (glycolysis, TCA cycle, pentose phosphate pathway), storage material pathways, amino acid
production, nucleotide pathways and product pathways (the biosynthesis of PenG). The biomass
composition of the glucose limited strain of Penicillium chrysogenum at a dilution rate of 0.5 hr-1 is
CH1.794N0.162O0.578P0.007S0.002-0.012.
In the above mentioned paper, the stoichiometric model serves as a basis for metabolic flux analysis,
which revealed that the penicillin biosynthesis requires major changes through the primary metabolic
pathways, caused by an increased demand for NADPH and the biosynthesis of β-lactam nucleus. Potential
bottlenecks for increasing the penicillin production are located around 4 principal nodes in the central
metabolism, which are located at g6P, 3PG, mitochondrial pyr and iCitr.
7
By letting the cells grown on different substrates in a carbon-limited culture, the metabolic fluxes through
the central metabolism can be manipulated but this had no effect on the penicillin biosynthesis, suggesting
that the central metabolism is not likely to be a potential bottleneck.
Subsequently the NADPH level has been decreased which resulting that the penicillin biosynthesis also
decreased. Hence based on metabolic flux analysis, the penicillin biosynthesis is limited by the supply and
regeneration of NADPH rather than the carbon precursors (Kleijn et al. 2007; van Gulik et al. 2000).
2.2 Background stoichiometric matrix and steady state rates
Once the metabolic reaction network has been defined that includes m metabolites and r reactions, the
stoichiometric matrix can be constructed containing m rows and r columns. The columns of the
stoichiometric matrix represent a chemical reaction and contains the stoichiometric coefficients of the
involved chemical species for that particular reaction.
Consider the following reaction scheme that contains two reactions that are close to equilibrium (note that
v2 & v4 are defined as net rates) and three dynamic rates and four intracellular metabolites:
v1
⎯⎯
→ X1
v2
v3
X 2 ⎯⎯
→ X3
v4
v5
X 4 ⎯⎯
→
Reaction Scheme 1
The stoichiometric matrix of this system is:
⎡1
⎢0
Sx = ⎢
⎢0
⎢
⎣0
−1
1
0
0
0⎤
−1 0 0 ⎥
⎥
1 −1 0 ⎥
⎥
0 1 −1⎦
0
0
(2.1)
Here the columns are organized as [v1 v2 v3 v4 v5] and the rows as [x1 x2 x3 x4].
Rank analysis indicates whether the matrix contains dependencies. For the matrix given in equation (2.1),
the rank is 4 and is said to be at full rank. If the rank is lower than the number of its components, the
matrix is called rank deficient and the dependencies should be removed from the matrix. The matrix in
(2.1) is underdetermined as the number of metabolites is smaller than the number of reactions and no
column rank deficiency is present. Hence additional equations are needed in order to get a closed system.
If the number of metabolites is higher than the number of reactions the system is over determined.
The steady state metabolic fluxes, written as J0, can be calculated by taking the mass balance, equation
(1.1) & (1.2) and setting dx/dt and dc/dt to zero, i.e.
0 = Sx ⋅ J0
(2.2)
0 = S c ⋅ J 0 + ( Din ⋅ c feed − Duit ⋅ c )
(2.3)
8
The steady state metabolic fluxes can be calculated by combining the above two equations and
multiplying the inverse of the stoichiometric matrix with the net conversion rate, denoted as qi.
−1
⎡S x ⎤ ⎡ 0 ⎤
⎡0⎤
J = ⎢ c ⎥ ⋅ ⎢ ⎥ = S −1 ⋅ ⎢ ⎥
⎣q i ⎦
⎣S ⎦ ⎣q i ⎦
0
(2.4)
Note that the net conversion rate of an intracellular metabolite is zero. The net conversion rate follows
from equation (2.3):
qi = − ( Din ⋅ c feed − Duit ⋅ c )
(2.5)
In order to calculate the inverse the combined stoichiometric matrix containing intracellular and
extracellular metabolites, denoted as S, must be square and possesses full rank.
2.3 The linlog format
In this project the reactions rates are expressed by the linlog format, which has been shown to be well
suited for metabolic reactions (Visser and Heijnen 2003). The linlog format has the following advantages:
•
Linear kinetic expression for reaction rate on kinetic parameters, i.e. the elasticities
•
Efficient parameter estimation, due to the linear dependency of its parameters.
•
Improved transferability between researches since dimensionless numbers are used.
•
Influence of metabolites easy incorporated, by setting the corresponding elasticity nonzero.
•
Many elasticities can be set to zero, which can improve the quality of estimation
Biochemical reaction rates depend on the concentrations of the involved metabolites and are further
assumed to be directly proportional to the enzyme activity.
v = e ⋅ f ( X1 ,..., X n )
(2.6)
0 0
Employing the Taylor expansion around a certain reference point (x , J ) yields
v
e
= 0
0
J
e
2
⎛ ∂v
⎞
∂2v
⋅ ⎜ i + ⋅ ( x − x0 ) + 2 ⋅ ( x − x0 ) + …⎟
∂x
⎝ ∂x
⎠
(2.7)
Subsequently this equation is divided by the reference points J0 and x0 and the higher order terms are
discarded.
v
e ⎛ x 0 ∂v ⎛ x
⎞⎞
≈
⋅ i + 0 ⋅ ⋅⎜ 0 − i⎟⎟
0
0 ⎜
J
e ⎝ J ∂x ⎝ x
⎠⎠
(2.8)
The final term can be locally assumed by taking the natural logarithm of the normalized concentration.
⎛ x
⎞
⎛ x⎞
⎜ 0 − i ⎟ ≈ ln ⎜ 0 ⎟
⎝x
⎠
⎝x ⎠
(2.9)
This assumption is only valid when the response of a metabolite remains relative close to its reference
conditions, as can been seen in Figure 2.2. Large perturbations in the system results in a large X/X0 ratio,
9
which causes that the above assumption may introduce a significant error. However due to homeostatis no
large metabolite perturbations are expected in the system. Nevertheless large perturbations of metabolite
concentrations, enzyme levels and fluxes are allowed (Visser et al. 2004a)
3
2
1
0
0
0.5
1
1.5
-1
2
2.5
3
3.5
4
X/ X0
-2
ln(X/X0)
(X/X0)-1
-3
Figure 2.2: Assumption made in equation (2.9)
This results in the following general equation for the reaction rate:
v
e⎛
⎛ x ⎞⎞
= 0 ⎜ i + Ex ⋅ ln ⎜ 0 ⎟ ⎟
0
J
e ⎝
⎝ x ⎠⎠
(2.10)
The normalized elasticity is defined as:
Ex =
x 0 ⎛ ∂v ⎞
⎜ ⎟
J 0 ⎝ ∂x ⎠
0
(2.11)
In the next chapter the elasticities are discussed in more detail.
2.4 Enzyme-metabolite connectivity
A priori knowledge about the interaction between metabolites and enzymes enhances the quality of the
estimation of the elasticities. The elasticity in question can be assigned as a non-zero entry in the elasticity
matrix if an interaction occurs between the enzyme and a metabolite. If no such interaction exists, the
elasticity is question should be set to zero.
Hence it is necessary to identify metabolites that are influencing the activity of the involved enzyme, i.e.
finding the connectivity between enzymes and metabolites. These metabolites can be substrates, products
or effectors, i.e. a molecule that influences the activity of the enzyme by binding at the regulatory site of
the protein which is different from the substrate-binding catalytic site.
Online databases such as (Brenda 2006; KEGG 2006; Metacyc 2006; SabioRK 2006), literature sources
and personal communication are used to determine the connectivity between enzymes and metabolites, the
mechanistic kinetic law equations, reversibility and if available the turnover number of the metabolites.
The kinetic law equations can help to identify which substrates and products are influencing the activity.
The literature search for metabolites that interact with the enzyme concerned focuses primary on the
genuses that belong in the family trichocomaceae. However if no information is available, information
10
obtained from other model organism such as the yeast, S. pombe and Neurocrassa crassa is used. A brief
overview of the fungi classification is presented below. The numbers between the brackets show the main
focus of the literature search.
Kingdom: Fungi
•
phylum: ascomycota
o
class: eurotiomycetes
ƒ
order: eurotiales
•
family: trichocomaceae (1)
o
o
genus: Penicillium, Aspergillus
class: saccharomycetes
ƒ
order: saccharomycetales
•
family: saccharomycetaceae (2)
o
genus:
Saccharomyces,
Candida,
Ashbya,
Kluyveromyces,
Yarrowia
o
class: schizosaccharomycetes
ƒ
order: schizosaccharomycetales
•
family: schizosaccharomycetaceae (2)
o
o
genus: Schizosaccharomyces
class: ascomycetes
ƒ
order: sordariomycete
•
family: sordariaceae (2)
o
genus: Neurospora
Nevertheless even with an extensive literature survey, there still remains uncertainty regarding the quality
and quantity of the information found. This is caused by:
•
the difficulty to translate information from experiments performed in vitro to a in vivo
environment, since many processes employed in a living cell are missing, which already have
been discussed in chapter 1.2.
•
not all metabolites have been tested for allosteric interactions. Experimental measurements may
be limited to a few metabolites which are likely to be effectors.
•
online databases may not contain all available data. For example the Brenda database does not
include t6P as an inhibitor for hexokinase despite that it has been reported in literature.
11
However the aim of the literature survey is to obtain as much kinetic information as possible, in order to
minimalize the uncertainty.
The stoichiometric model presented in (van Gulik et al. 2000) consists of many reactions that are the sum
of several intermediate catalyzed conversions. This suggests that several enzymes are involved in
catalyzing one single reaction as defined in the stoichiometric model. In a metabolic network many
reactions are near equilibrium and their reaction rate is effectively controlled by varying the concentraions
of the involved reactants and products and do not require the postulation of kinetic rate expressions.
However certain enzymatic reactions, such as phosphofructokinase, operate far from equilibrium and are
targets for metabolic regulation by feedback inhibition or allosteric interaction. Changes in substrate level
have relatively little effect on the rate as the enzyme is close to saturation. The rate can be enhanced by for
example allosteric interaction. These irreversible reactions are highly exergonic, i.e. the free energy
ΔG<<0. In each metabolic pathway there is an irreversible reaction that commits its product to move
down the pathway. The free energy can be calculated by:
ΔG = ΔG 0 + R ⋅ T ⋅ ln ( K eq )
(2.12)
Here ΔG0 is the free energy at reference conditions, R the gas contant, T the temperature and Keq the mass
action constant. For reactions that are close to equilibrium ΔG = 0 as the free energy of the forward
reaction balances that of the backward reaction.
Irreversible reactions are the rate determining step in the pathway and require the proposal the kinetic rate
expression. With the help of online database, a preliminary selection of the dynamic reactions can be
made for which a kinetic expression should be proposed. In case of a lumped reaction, the reaction
kinetics is equal to the kinetics of the irreversible reaction as it is the limited reaction rate in the lumped
reaction.
2.5 Model Simplification
The stoichiometric model can be simplified by eliminating redundant compounds such as conserved
moieties. It is assumed that the sum of these conserved moieties remains constant. The dynamics of a
single metabolite can be expressed as a function of the total and the others:
X 1 + X 2 + ... + X n = Σ
(2.13)
in which Σ denotes the total amount of the conserved moiety and Xi the individual metabolite
concentrations. The above equation can also be rewritten into its differential form:
dX 1 dX 2
dX n
+
+ ... +
=0
dt
dt
dt
(2.14)
12
Conserved moieties can be identified by rank analysis of the stoichiometric matrix. The number of
conserved moieties relations, nCM can be found by
nCM = m − Rank(S x )
(2.15)
in which m denotes the number of metabolites and Sx the stoichiometric matrix containing intracellular
metabolites. The individual species that hold the conservation principle can be found by taking the rational
basis of the null space of the transpose of Sx, written as Lcm, which calculates the set of solutions of the
homogenous relations shown in equation (2.2).
The ncm dependent concentrations can be determined by using the following matrix multiplication:
dx cm
Τ
= ( L cm ) ⋅ S x ⋅ v
dt
(2.16)
However since the linlog format is used a simpler correlation among the conserved moieties can be
derived. Subtracting and subsequently diving equation (2.13) by the reference conditions yields the
following relation:
⎞ ⎛Σ
⎞
X 10 ⎛ X 1
X n0 ⎛ X n
⎞
−
+
+
− 1⎟ = ⎜ 0 − 1⎟
1
...
⎟
0 ⎜
0
0 ⎜
0
Σ ⎝ X1
Σ ⎝ Xn
⎠
⎠
⎠ ⎝Σ
(2.17)
Recall the assumption made in equation (2.9) which leads to the following simplification:
⎛ X ⎞ ∑0 ⎛ ∑ ⎞
X0 ⎛ X ⎞
ln ⎜ 10 ⎟ = 0 ln ⎜ 0 ⎟ − ... − n0 ln ⎜ n0 ⎟
X1 ⎝ X n ⎠
⎝ X1 ⎠ X1 ⎝ ∑ ⎠
(2.18)
2.6 Model Reduction
“A designer knows he has achieved perfection not when there is nothing left to add, but when there is
nothing left to take away,” Antione de Saint-Exupry.
In this chapter the possibilities for the reduction of the model will be elaborated. Model reductions aim to
decrease the number of metabolites, rates and parameters to be estimated which reduces the computational
time. Furthermore a reduced system allows a better focus on the most important interactions and limiting
rates.
2.6.1 Data-driven reduction of metabolic network
13
Due to that time dependent responses have been measured for a limited number of metabolites, a datadrive reduction of the metabolic network can be applied, which eliminates the unmeasured metabolite by
summing the involved reactions. These unmeasured metabolites have not been measured due to their low
concentrations or because their concentration can be determined from a pseudo equilibrium reaction, see
next chapter. In case the concentration is too low to measure, the metabolite can be assumed to be at
pseudo steady state, see chapter 2.6.6.
Since only the average cell concentration have been measured, it is not possible to determine the
concentrations within the three compartments individually. No concentration ratio between these
compartments has been found for Penicillium chrysogenum and hence the model is reduced to one
compartment.
2.6.2 Identification of PEQ reactions
The system can be further reduced by identifying the reactions that are close to equilibrium. In Table 2.1
the candidates of reactions that could be near equilibrium are listed, which initially is based on the
reversibility of the reactions, as listed in appendix C. Irreversible reactions are frequently the rate
determining step at which the pathway is regulated and hence are not likely to be near equilibrium.
However this does not imply that every reaction which is reported to be reversible is close to equilibrium
at the time window of observation.
Table 2.1: Candidates that can be PEQ
Glycolysis
1
r1.2
g6P = f6P
2
r1.4
f16P = 2 GAP
3
l1.2
GAP+NAD+Pi+ADP = 3PG + ATP + H + NADH
4
l1.3
3PG = H2O + PEP
Pentose phosphate pathway
5
r2.3
Ribu5P = Rib5P
6
r2.4
Ribu5P = Xylu5P
7
r2.5
Rib5P + Xylu5P = GAP + sed7P
8
r2.6
GAP + sed7P = E4P + f6P
9
r2.7
E4P + Xylu5P = f6P + GAP
TCA cycle
14
10
r4.2
AcCoA + H2O + OAA = citr + H + HCoA
11
r4.3
citr = iCitr
12
r4.8
ADP + Pi + succCoA = ATP + HCoA + succ
13
r4.9
FAD + succ = FADH2 + fum
14
r4.10
fum + H2O = mal
15
r4.11
mal + NAD = H + NADH + OAA
Anaplerotic pathways
16
r5.1
ATP + CO2 + H2O + pyr = ADP + 2 H + OAA + Pi
Transport of 1-C Compounds
17
r8.1
gly + NAD + THF = CO2 + METHF + NADH + NH4
18
r8.2
H + METHF + NADH = MYTHF + NAD
Transport across plasma membrane
19
r9.1
ATP+H2O = ADP+H+Pi
20
r9.6
O2:ext = O2:cyt
21
r9.7
CO2:cyt = CO2:ext
22
r9.15
H2O:cyt = H2O:ext
Amino Acid synthase
23
r11.1
aKG + H + NADPH + NH4 = glu + H2O + NADP
24
r11.2
ATP + glu + NH4 = ADP + gln + H + Pi
25
r11.10
ser + THF = gly + H2O + METHF
26
r11.16
glu+OAA = aKG + asp
27
r11.20
homcys + MYTHF => met + THF
28
r11.22
glu + pyr = aKG + ala
Transfer of 1C Compounds
29
r13.6
ATP+H2O+gln+UTP=> ADP+Pi+CTP+ 2H + gln
Synthesis of glycogen and polysaccharides
30
l17.3
g6P = g1P
In order to check whether a certain reaction is close to equilibrium, the mass action ratio of the reactions
listed in Table 2.1 is calculated as a function of time. If the mass action ratio remains approximately
constant during the pulse experiment, the reaction can be assumed to be near equilibrium.
15
2.6.3 Calculation of unmeasured metabolites from PEQ rates.
However since not all necessary metabolites have been measured, e.g. NADH, NADPH, THF etc, it is
necessary to assume that certain reactions are close to equilibrium. In order to calculate the concentrations
of the species involved in the non-oxidative branch of the PPP it is assumed that reactions r2.3-r2.7 are
close to equilibrium. Furthermore it is assumed that part of the glycolysis, f16P till 3PG forms an
equilibrium pool so that the NADH/NAD ratio can be calculated under the assumption of Pi = 10 mM.
Another option to calculate the NADH/NAD ratio is the equilibrium pool of aspartate transmutase, malate
dehydrogenase, and fumerase, although less data points are available, Since an equilibrium pool of C4
metabolites in the TCA cycle exists (Nasution et al. 2006), the profiles of FADH2/FAD, OAA and
succCoA can be determined. Furthermore it is assumed that the citrate (Si)-synthase and aconitrate
hydratase are close to equilibrium so that the species AcCoA/HCoA, citr and iCitr can be calculated.
The NADPH/NADP profile can be calculated based on glutamate dehydrogenase (r11.1), which is
supposed to be near equilibrium but requires the additional assumption of the NH4 concentration. In
addition it is assumed that the serine hydroxymethyltransferase (r11.10) and N5-N10-methylene-THF
reductase (r8.2) are close to equilibrium so that the THF species can be determined. N5-N10-methyleneTHF reductase has a large equilibrium constant of 4.5*1010 and as a consequence a high Gibbs-energy,
suggesting that it might be an irreversible reaction subject to regulation, despite the fact that is has been
reported as highly reversible (Voet and Voet 2004). Other reactions that involve THF species are the
methionine synthase (r11.20) and glycine cleavage system (r8.1) which supposed to be irreversible
(KEGG 2006) and for which no equilibrium constants have been found in literature or any databases
(NIST 2007). In the next chapters the calculation of these unknown metabolites is discussed.
2.6.3.1 Glycolysis metabolites
The reactions catalyzed by aldolase and triophosphate isomerase have been lumped and will be assumed
to be near equilibrium. The equilibrium constant is defined as
2
GAP ]
[
K r1.4 =
[ f 16 P ]
(2.19)
Hence the GAP concentration, required for the calculation of a few unknown metabolites, is equal to:
[GAP ] = ( K r1.4 ⋅ [ f 16 P ])
0.5
(2.20)
The NADH/NAD ratio can be determined from reaction r1.5 which is catalyzed by glyceraldehyde-3phosphate. The necessary equilibrium constants are defined as:
16
K r1.5 =
[13PG ] ⋅ [ NADH ]
[GAP ] ⋅ [ Pi ] ⋅ [ NAD ]
[3PG ] ⋅ [ ATP ]
[13PG ] ⋅ [ ADP ]
[2 PG ]
K r1.7 =
[3PG ]
K r1.6 =
(2.21)
(2.22)
(2.23)
Table 2.2: Equilibrium constants & assumed concentrations
Symbol
Value
Equilibrium constants
Kr1.4
2.545*10-6 M
(Veech et al. 1969)
Kr1.5
0.487
(Cornell et al. 1979)
Kr1.6
2.27*103
(Cornell et al. 1979)
Kr1.7
0.0920
(Guynn, R.W. et al., 1982)
Kr2.3
0.833
(Casazza and Veech 1986)
1.82
(Casazza and Veech 1986)
Kr2.4
Kr2.5
0.48
(Casazza and Veech 1986)
Kr2.6
0.37
(Casazza and Veech 1986)
Kr2.7
0.0337
(Casazza and Veech 1986)
Kr4.2
2.24*106
(Guynn et al. 1973)
Kr4.3
0.041
(Veloso et al.,1973)
0.949
(Lynn and Guynn 1978)
Kr4.8
Kr4.9
0.85
http://people.unt.edu/~hds0006/tca/index.htm
Kr4.11
2.41E-5 M
(Guynn et al. 1973)
Kr8.2
4.5*1010
(Wohlfarth and Diekert 1991)
Kr11.1
9.886*10-7
(Engel and Dalziel 1967)
10.72
(Schirch et al. 1977)
Kr11.10
Henry constants
3.4*10-2 M/atm
HCO2
(Sander 1999)
1.3*10-3 M/atm
HO2
(Sander 1999)
Conserved moieties sums
∑NAD
1 mM
assumption
∑NADP
1 mM
assumption
∑FAD
1 mM
assumption
∑CoA
1 mM
assumption
∑THF
1 mM
assumption
Assumptions concentration
10 mM
[Pi:cyt]
assumption, as in (Teusink et al. 2000)
[NH4:cyt] 0.1 mM
assumption
[SO4:cyt] 1 mM
assumption
10-7 M
[H:cyt]
pH = 7, assumption
1 mM
[arg:cyt]
assumption
Since the sum of 2PG and 3PG has been measured the following holds:
[2 PG ] + [3PG ] = ∑r1.7
(2.24)
17
Furthermore due to the fact that NAD/NADH is a conserved moiety the following holds:
[ NADH ] + [ NAD ] = ∑ NAD
(2.25)
The constant ∑NAD has not been determined and it will be assumed, see Table 2.2. Moreover the
intracellular concentration of Pi and the pH has not been measured and will also need to be assumed.
Solving for 3PG, NADH and NAD yields
[3PG ] =
∑r1.7
K r1.7 + 1
[ NADH ] =
(1 + K r1.7 ) ⋅ ∑ NAD
[ ATP ] ⋅ ∑r1.7
1 + K r1.7 +
K r1.5 ⋅ K r1.6 . [ ADP ] ⋅ [GAP ] ⋅ [ Pi ]
[ NAD ] = CNAD − [ NADH ]
(2.26)
(2.27)
(2.28)
2.6.3.2 PPP metabolites
The unmeasured metabolites with respect to the non-oxidative pentose phosphate pathway are Ribu5P,
Rib5P, Xylu5P, E4P and sed7P. Recall that all the reactions in the non-oxidative part the pentose
phosphate pathway are taken as near equilibrium. As a consequence these metabolites can be written as a
function of the measured f16P and f6P. The required equilibrium constants are defined as:
K r 2.3 =
[ Ribu5P ]
[ Rib5P ]
(2.29)
K r 2.4 =
[ Xylu5P ]
[ Ribu5P ]
(2.30)
K r 2.5 =
[ Rib5P ] ⋅ [ Xylu5P ]
[GAP ] ⋅ [ sed 7 P ]
(2.31)
K r 2.6 =
[ E 4P] ⋅ [ f 6P]
[GAP ] ⋅ [ sed 7 P ]
(2.32)
K r 2.7 =
[ E 4 P ] ⋅ [ Xylu5P ]
[GAP ] ⋅ [ f 6 P ]
(2.33)
The relevant results are:
18
[ Xylu5P ] =
K r 2.7 ⋅ [ f 6 P ]
⎛
⎞
K r 2.7 ⋅ [ f 6 P ]
K r 2.6 ⋅ ⎜ [GAP ] ⋅
⎟
2
K r1.4 ⋅ K r 2.3 ⋅ K r 2.4 ⋅ K r 2.5 ⋅ K r 2.6 ⋅ [ f 16 P ] ⎠
⎝
2
[ Rib5P ] =
[ E 4P] =
1
3
[ Xylu5P ]
K r 2.3 ⋅ K r 2.4
K r 2.7 ⋅ [GAP ] ⋅ [ f 6 P ]
[ Xylu5P ]
(2.34)
(2.35)
(2.36)
2.6.3.3 TCA metabolites
OAA is synthesized by malate dehydrogenase and this reaction is assumed to be near equilibrium. The
corresponding equilibrium constant is defined as:
K r 4.11 =
[OAA] ⋅ [ NADH ]
[mal ] ⋅ [ NAD ]
(2.37)
Hence the OAA concentration is equal to:
[OAA] =
K r 4.11 ⋅ [ NAD ] ⋅ [ mal ]
[ NADH ]
(2.38)
The sum of the metabolites citr and iCitr have been measured, hence the following holds
[iCitr ] + [ citr ] = ∑citr
(2.39)
The reaction catalyzed by aconitate hydratase will be taken as near equilibrium and the corresponding
equilibrium constant is defined as:
K r 4.3 =
[iCitr ]
[ citr ]
(2.40)
Hence the citr concentration equals:
[citr ] =
∑citr
K r 4.3 + 1
(2.41)
The concentrations of the CoA species can be determined from the near equilibrium reactions that are
catalyzed by citrate synthase (r4.2), succinate-CoA ligase (r4.8) and the CoA conversed moiety relation.
The corresponding equilibrium constants are defined as follows:
19
K r 4.2 =
[ citr ] ⋅ [ HCoA]
[OAA] ⋅ [ AcCoA]
(2.42)
K r 4.8 =
[ succ] ⋅ [ ADP ] ⋅ [ HCoA]
[ ATP ] ⋅ [ Pi ] ⋅ [ succCoA]
(2.43)
HCoA + succCoA + AcCoA = ∑CoA
(2.44)
The constant ∑CoA has not been determined and will be assumed, see Table 2.2 Solving these equations for
the CoA species yields:
[ AcCoA] =
[ HCoA] =
∑CoA
[OAA] ⋅ ⎛ 1 + [ succ ] ⋅ [ ADP ] ⎞
1 + K r 4.2 ⋅
[ citr ] ⎜⎝ K r 4.8 ⋅ [ ATP ] ⋅ [ Pi ] ⎟⎠
K r 4.2 ⋅ [ AcCoA] ⋅ [OAA]
[ citr ]
[ succCoA] = ∑CoA − [ AcCoA] − [ HCoA]
(2.45)
(2.46)
(2.47)
The concentrations of FADH2 and FAD can be calculated by the following equations:
K r 4.9 =
[ fum ] ⋅ [ FADH 2]
[ succ] ⋅ [ FAD ]
[ FADH 2] + [ FAD ] = ∑ FAD
(2.48)
(2.49)
The value of the constant ∑FAD has not been determined and will be assumed, see Table 2.2. Rearranging
yields
[ FAD ] =
∑ FAD
[ succ ]
1 + K r 4.9 ⋅
[ fum]
[ FADH 2] = ∑ FAD − [ FAD ]
(2.50)
(2.51)
2.6.3.4 NADPH/NADP concentrations
The NADPH and NADP concentration can be determined from the near equilibrium reaction catalyzed by
glutamate dehydrogenase (r11.1) and the conserved moiety relation. The equilibrium constant of the
glutamate dehydrogenase reaction is defined as:
20
K r11.1 =
[ aKG ] ⋅ [ NH 4] ⋅ [ NADPH ]
[ glu ] ⋅ [ NADP ]
[ NADPH ] + [ NADP ] = ∑ NADP
(2.52)
(2.53)
Eliminating NADP results in:
NADPH =
∑ NADP
aKG
[ ] ⋅ [ NH 4]
1+
K r11.1 ⋅ [ glu ]
[ NADP ] = ∑ NADP − [ NADPH ]
(2.54)
(2.55)
2.6.3.5 THF species
The THF species can be calculated as the reactions catalyzed by methylenetetrahydrofolate reductase
(r8.2) and glycerine hydroxymethyltransferase (r11.10) can be considered as near equilibrium. The
corresponding equilibrium constants are defined as:
K r 8.2 =
[ MYTHF ] ⋅ [ NAD ]
[ METHF ] ⋅ [ NADH ]
(2.56)
[ gly ] ⋅ [ METHF ]
[ ser ] ⋅ [THF ]
(2.57)
K r11.10 =
Furthermore since the THF species are a conserved moiety, the following holds:
[THF ] + [ MYTHF ] + [ METHF ] = ∑THF
(2.58)
Rearranging yields:
[THF ] =
∑THF
[ ser ] ⋅ ⎛1 + K [ NADH ] ⎞
1 + K r11.10 ⋅
r 8.2
[ gly ] ⎜⎝
[ NAD ] ⎟⎠
[ METHF ] = K r11.10 ⋅
[ ser ] ⋅ [THF ]
[ gly ]
(2.59)
(2.60)
2.6.3.6 O2/CO2
The dissolved extracellular CO2 can be calculated according to:
qCO 2 = kla ⋅ (CCO 2,l − CCO 2,l * )
(2.61)
21
CCO 2,l * = H CO 2 ⋅ pCO 2 ⋅ Ptot
(2.62)
in which qCO2 is the calculated net conversion rate, HCO2 is the Henry constant for CO2, pCO2 the measured
partial pressure and Ptot the total pressure. Rearranging yields the dissolved CO2 concentration:
CCO 2,l = H CO 2 ⋅ pCO 2 ⋅ Ptot +
qCO 2
kla
(2.63)
The dissolved extracellular oxygen can be calculated according to
qO 2 = − kla ⋅ (CO 2,l − CO 2,l * )
(2.64)
CO 2,l * = H O 2 ⋅ pO 2 ⋅ Ptot
(2.65)
where qO2 is the calculated net conversion rate, HO2 is the Henry constant for O2, pO2 the measured partial
pressure and Ptot the total pressure. Rearranging yields the dissolved CO2 concentration:
CO 2,l = H O 2 ⋅ pO 2 ⋅ Ptot −
qO 2
kla
(2.66)
It is assumed that the diffusion of O2 and CO2 across the plasma membrane is relatively fast compared to
its consumption rate and hence the extracellular concentration equals the intracellular concentration. The
transient behaviour of O2 and CO2 follows from the corresponding measured partial pressure.
2.6.3.7 Miscellaneous metabolites
Despite the above PEQ relations there are still a few metabolites that have not been determined, e.g. PAA,
penG, arg, tre, psacch and ExtPept.
The extracellular concentration of PAA, penG, tre, psacch and ExtPept can be determined from the mass
balance from the reactions. The mass balance for last four metabolites is:
[ X i ]ext =
qi ⋅ C x
Fout
(2.67)
in which the effluent flow rate is denoted as Fout [l/s], qi the biospecific net conversion rate of component i
(μmol/gDW/s) and Cx biomass concentration (gDW/l). Rearranging the mass balance over the fermentor
yields the extracellular concentration of PAA.
[ X PAA ]ext =
Fin
q ⋅C
⋅ X PAA, feed − PAA x
Fuit
Fout
(2.68)
According to the original stoichiometric model (van Gulik et al. 2000), the transport across the plasma
membrane is driven by the concentration gradient, i.e. passive transport. It will be assumed that the
concentrations of PAA, penG and ExtPept remain constant during the time window of observation. The
concentration ratio between the extracellular and cytosol concentration of PAA, penG, ExtPept and psacch
22
is unknown and will be assumed to be 4. The intracellular concentration of tre has been measured and is
17 mM [40 μmol/gDw].
The dynamic perturbation experiment has shown an increased production of storage materials (Nasution
2007) and as a consequence the tre and psacch can not be considered to constant. The unmeasured
metabolites arg will be assumed to remain constant at 1mM.
2.6.4 Time scale analysis
Time scale analysis can be used to further reduce the model as many reactions occur at different time
scales, for which the following distinction can be made:
•
Pseudo equilibrium relations (PEQ)
•
Pseudo steady state relations (PSS)
•
Dynamic relations
•
Frozen relations
These relations can be found by comparing the turnover times of a reaction, written as τ, to the time
window of observation, τobs. The turnover time can be calculated according to:
τ=
X0
J in0
(2.69)
Here X0 is the steady state concentration, and Jin0 denotes the net influx which can be calculated by:
J 0in = S in ⋅ J 0
(2.70)
in which Sin is the stoichiometric matrix Sx where all negative stoichiometric coefficients are set to zero. J0
is a column vector containing the metabolic fluxes at steady state. Note that if only the average cell
concentration is known, the transport steps across membrane to or from inner compartments should be
omitted from Sx in order to avoid double counting of metabolic fluxes.
Frozen concentrations can be assumed to be constant during the time window of observation as their
turnover time is much larger than the window of observation. If the turnover time is much smaller than the
time window of observation, it can be identified as a PEQ or PSS, which is discussed in chapter 2.6.5 and
2.6.6. In Table 2.3 the time scales of these relations are listed.
23
Table 2.3: Time scale separation
Time scale
<<τobs
<τobs
τobs
>>τobs
Rates
v=∞
Spss*v=0
v=f([x])
v=0
Assumption
PEQ
PSS
dynamic
frozen
Abbreviations: PEQ = pseudo equilibrium relation, PSS = pseudo steady state relation, τobs = time window of observation
These relations can be used to reduce the number of independent compounds and reactions, which reduces
the computational time and enhances the insight of the behaviour of the system.
2.6.5 Pseudo Equilibrium (PEQ)
A reaction which is supposed to be in pseudo equilibrium is characterized by:
•
A much smaller timescale compared to the time window of observation.
•
Conversion rate of the forward and backward reactions are significantly higher than the net
conversion rate.
•
The mass action ratio remains constant during the time window of observation.
Consider the following reaction scheme:
a[A] + b[B]
v1
v2
c[C] ⎯⎯
→ [ D]
Reaction Scheme 2
Here the conversion of A and B into C is assumed to be near equilibrium, i.e. is a PEQ. The mass action
ratio is:
(C )
a
b
( A) ⋅ ( B )
(C )
and at steady state
( A ) ⋅(B )
0 c
c
=K
0 a
0 b
=K
(2.71)
Dividing by its steady state values and taking the logarithm yields:
T
⎡
A
B
C ⎤
[ a b c ] ⋅ ⎢ln ⎛⎜ 0 ⎞⎟ ln ⎛⎜ 0 ⎞⎟ ln ⎛⎜ 0 ⎞⎟ ⎥ = 0
⎣ ⎝ A ⎠ ⎝ B ⎠ ⎝ C ⎠⎦
(2.72)
This linear relation in the logarithmic space is used to remove equilibrium pools from the dynamic model,
i.e. metabolite C is combined with A & B to a new equilibrium pool and the near equilibrium reaction v1
disappears.
Now let’s consider that in reaction scheme 2, the metabolic flux v2 is dependent by metabolite C, i.e.
⎛
⎛ C ⎞⎞
v2 = J 20 ⋅ ⎜ i + ε 2,C ⋅ ln ⎜ 0 ⎟ ⎟
⎝ C ⎠⎠
⎝
(2.73)
24
Elimination of ln(C/C0) with equation (2.72) into equation (2.73) yields:
⎛
⎧a
⎛ A ⎞ b ⎛ B ⎞⎫ ⎞
v2 = J 20 ⋅ ⎜ i + ε C ,2 ⋅ ⎨ ⋅ ln ⎜ 0 ⎟ + ⋅ ln ⎜ 0 ⎟ ⎬ ⎟
⎝A ⎠ c
⎝ B ⎠⎭ ⎠
⎩c
⎝
(2.74)
This linear logarithmic dependency of the involved metabolites in a reaction assumed to be at PEQ, allows
the removal of one metabolite from the ODE system. The advantage of the PEQ relation is that it produces
a simpler from than the PSS relation as it contains less parameters, see chapter 2.6.6.
In this chapter a systematic approach is discussed to derive linear logarithmic relations between
metabolites that are involved in a reaction close to equilibrium.
Recall reaction scheme 1 in which reaction 2 is very fast and can be considered to be close to equilibrium
(reaction 4 is not considered for the moment to simplify the discussion). In this case the rate of reaction 2
can be eliminated from the differential equations by summing the differential equations of the metabolites
1 and 2, i.e.
dX 1
dX 2
= v1 − v2 +
= v2 − v3
dt
dt
(2.75)
yields
dp1
= v1 − v3
dt
(2.76)
in which the new defined equilibrium pool p1 is sum of X1 and X2. The individual concentrations of X1
and X2 can be calculated by the pool equation and the equilibrium equation:
p1 = [ X 1 ] + [ X 2 ]
K=
[X2]
[ X1 ]
(2.77)
(2.78)
The full stoichiometric matrix, written as S (mxr) is arranged in such a way like shown in Figure 2.3. The
columns that contain the net rates corresponding to PEQ, vpeq, are organized on the left side of the matrix
and the involved metabolites on the top, written as xpeq. The submatrix that contains the PEQ rates and the
involved metabolites is denoted as Speq.
25
vpeq
vdyn
xpeq
Speq
S2
xdyn
S3
S4
Figure 2.3: Arrangement stoichiometric matrix S
The pool composition matrix, written as P and containing the pool equations such as in equation (2.77),
can be determined by calculating the rational basis of the null space of SpeqT, denoted as Lpeq.
PT = null ( S peq Τ , ' r ' ) = L peq
(2.79)
Note that null(SpeqT,’r’) is written as a Matlab command for clarity sake. The ‘r’ refers to the calculation of
the rational basis of the nullspace. The rational basis of the null space puts the matrix is a much more
comprehensive form than the nullspace on an orthonormal basis. This avoids the calculation of the
transformation matrix T as presented in (Visser et al. 2000). In the paper the transpose of the nullspace is
calculated by multiplying this transformation matrix with the null space of SpeqT. The result is however
similar than presented in equation (2.79).
Equilibrium pools are defined as invariant sums within PEQ reactions and hence the following holds:
0 = P ⋅ rpeq
(2.80)
Here, the net conversion rates of the pseudo equilibrium reactions are denoted as rpeq, which can be
calculated by multiplying Speq with vpeq.
0 = P ⋅ S peq ⋅ v peq
(2.81)
This holds for every pseudo equilibrium rate, and hence the following also holds:
0 = P ⋅ S peq or 0 = P ⋅ ( S peq )
Τ
(2.82)
Hence P lies in the null space of SpeqT.
The negative entries in the null space Lpeq are removed by summation of the columns. Subsequently the
pseudo equilibrium rates and metabolites are eliminated from the stoichiometric matrix whereas the
equilibrium pools are introduced which can be done by the following transformation:
S p = ( L peq ) ⋅ S v _ peq
Τ
(2.83)
26
The submatrix Sv_peq consists of Speq and S2, shown in Figure 2.3. The equilibrium pool balances are
given by Sp. The reduced matrix can be obtained by replacing Sv_peq with Sp which results that the columns
that represent the PEQ rates contain zeros and can subsequently be eliminated from the stoichiometric
matrix. The individual concentrations within the equilibrium pool can be calculated from the pool
equations and the equilibrium equations.
This is further elaborated in Example 2.1.
Example 2.1
Recall reaction scheme 1, which consist of two PEQ rates.
v1
⎯⎯
→ X1
v2
v3
X 2 ⎯⎯
→ X3
v4
v5
X 4 ⎯⎯
→
Reaction Scheme 1
The stoichiometric matrix has been given in equation (2.1). First the reduced stoichiometric matrix is first
derived manually after which the above discussed mathematical procedure is discussed. The following
equilibrium pools are created:
p1 = [ X 1 ] + [ X 2 ] for which the mass balance is
dp1
= v1 − v3
dt
p2 = [ X 3 ] + [ X 4 ] for which the mass balance is
dp2
= v3 − v5
dt
Hence the reduced stoichiometric model, written as Sred, of this system is:
−1 0 ⎤
1 −1⎥⎦
⎡1
S red = ⎢
⎣0
Here the rows are organized as [p1 p2] and the columns as [v1, v3, v5].
The subsystem of the pseudo equilibrium rates and corresponding metabolites, Speq is:
S peq
⎡ −1 0 ⎤
⎢1 0⎥
⎥
=⎢
⎢ 0 −1⎥
⎢
⎥
⎣0 1⎦
Multiplying the rational basis of the null space of the transpose of Speq with [Speq S2] yields:
⎡ 1 1 0 0⎤
S 2 ⎤⎦ = ⎢
⎥ ⋅ ⎡⎣S peq
⎣0 0 1 1⎦
1 −1 0 ⎤
0 1 −1⎥⎦
S p = ( L peq ) ⋅ ⎡⎣S peq
Τ
⎡ 0
Sp = ⎢
⎣ 0
0
0
S 2 ⎤⎦
27
Since all metabolites in the full stoichiometric matrix S, are involved in all PEQ reactions, the
stoichiometric matrix can be replaced by Sp. Elimination of the PEQ rates yields the reduced
stoichiometric matrix, Sred.
⎡1
S red = ⎢
⎣0
−1 0 ⎤
1 −1⎥⎦
From the pool matrix, i.e. LpeqT, the equilibrium pool relations can be found. It can be shows that:
⎡ x1 ⎤
⎢ ⎥
p1 = x1 + x2
⎡ p1 ⎤ ⎡ 1 1 0 0⎤ ⎢ x2 ⎥
=
⋅
⎢ p ⎥ ⎢0 0 1 1⎥ ⎢ x ⎥ ⇔ p = x + x
⎦
2
3
4
3
⎣ 2⎦ ⎣
⎢ ⎥
⎣ x4 ⎦
2.6.6 Pseudo Steady State (PSS)
After the identification of conserved moieties and PEQ rates the model can be further reduced by
removing the PSS relations from the ODE system.
The general mass balance given by equation (1.1) and introducing the linlog kinetic format given by
(2.10) can be arranged into
τ
d ( x x0 )
dt
( )
= J 0in
−1
⋅ S ⋅ J0 ⋅
e⎛
⎛ x c ⎞⎞
i + Ex,c ⋅ ln ⎜ 0 , 0 ⎟ ⎟
0 ⎜
e ⎝
⎝ x c ⎠⎠
(2.84)
in which Jin0 is the net influx, J0 the steady state metabolic flux, Ex,c the elasticities and x & c the internal
and external metabolites concentration. Note that the superscript 0 denotes the steady state condition.
For pseudo steady state metabolites the turnover time is very small and as a consequence the ODE system
is converted to a DAE system, see equation (2.85). The rates of production and consumption of pseudo
steady state metabolites must be balanced, and as a consequence the rates of pss rates are not independent.
⎛
⎛ x c ⎞⎞
S pss ⋅ J 0 ⋅ ⎜ i + E x ,c ⋅ ln ⎜ 0 , 0 ⎟ ⎟ =0
⎝ x c ⎠⎠
⎝
(2.85)
Here Spss denotes the npss x r stoichiometric matrix in which npss is the number of pss metabolites and r the
number of reactions. The elasticity matrix (rxn) and the concentration profile (nx1) remains at its original
size. Since SpssJ0i is equal to zero the above equation simplifies into:
28
⎛ x c⎞
S pss ⋅ J 0 ⋅ Ex ⋅ ln ⎜ 0 , 0 ⎟ =0
⎝x c ⎠
(2.86)
Let’s consider the following reaction scheme, which is the oxidative branch of the pentose phosphate
pathway:
vr 2.1
vr 2.2
g 6 P ⎯⎯⎯
→ 6 PGluct ⎯⎯⎯
→ Rib5P
Reaction Scheme 3
The reactions are denoted according to the notation used in (van Gulik et al. 2000). Reaction r2.1 is
activated by g6P and inhibited by ATP and NADPH. In addition reaction r2.2 is activated by 6PGluct and
inhibited by ATP and NADPH (Vaseghi et al. 2001). Hence the following holds:
⎡
⎛
⎛ g 6P ⎞
⎛ NADPH ⎞
⎛ ATP ⎞ ⎞ ⎤
0
+ ε r 2.1, NADPH ⋅ ln ⎜
+ ε r 2.1, ATP ⋅ ln ⎜
⎢ J r 2.1 ⋅ ⎜ i + ε r 2.1, g 6 P ⋅ ln ⎜
⎥
0 ⎟
0 ⎟
0 ⎟⎟
⎝ NADPH ⎠
⎝ ATP ⎠ ⎠ ⎥
⎝ g 6P ⎠
⎝
⎢
[ −1 1] ⋅ ⎢
⎥=0
⎛
⎞
⎛
⎞
6
Pgluct
NADPH
ATP
⎛
⎞
⎛
⎞
⎢ J r02.2 ⋅ ⎜ i + ε r 2.2,6 Pgluct ⋅ ln ⎜
⎥
+ ε r 2.2, NADPH ⋅ ln ⎜
+ ε r 2.2, ATP ⋅ ln ⎜
0 ⎟
0 ⎟
0 ⎟⎟
⎢⎣
6
Pgluct
NADPH
ATP
⎝
⎠
⎝
⎠
⎝
⎠
⎝
⎠ ⎥⎦
(2.87)
0
since Jr2.2 =
Jr2.10,
rearranging yields:
⎛ 6 Pgluct ⎞
⎛ g 6P ⎞
⎛ ATP ⎞
⎛ NADPH ⎞
ln ⎜
= α1 ⋅ ln ⎜
+ α 2 ⋅ ln ⎜
+ α 3 ⋅ ln ⎜
0 ⎟
0 ⎟
0 ⎟
0 ⎟
⎝ ATP ⎠
⎝ NADPH ⎠
⎝ 6 Pgluct ⎠
⎝ g 6P ⎠
(2.88)
in which
α1 =
ε r 2.1, g 6 P
(ε r 2.1, ATP − ε r 2.2, ATP ) ;α = (ε r 2.1,NADPH − ε r 2.2,NADPH )
;α 2 =
3
ε r 2.2.6 Pgluct
ε r 2.2.6 Pgluct
ε r 2.2.6 Pgluct
(2.89)
As already mentioned in chapter 2.6.5, the PSS relation contains much more parameters than the PEQ
relation. Note that the individual elasticities, e.g. εr2.1,ATP or εr2.2, NADPH, can not be determined individually.
In case 6Pgluct is an effector for a reaction, equation 2.26 is used to eliminate 6Pgluct.
The kinectic parameters αi, can be determined by linear regression for which the equation is written in
matrix form:
⎡α1 ⎤
y = X ⋅ ⎢α 2 ⎥ = X ⋅ b
⎢ ⎥
⎢⎣α 3 ⎥⎦
(2.90)
in which X is a dpxn matrix contain the data points (dp), y a dpx1 vector containing the measured values
of 6Pgluct. The three unknown parameters can be estimated by linear regression:
b = (XT ⋅ X ) ⋅ XT ⋅ y
−1
(2.91)
Matrix X requires the same amount of data points for each metabolite.
29
2.6.6.1 Removing pss metabolites from stoichiometric model
Let S be the stoichiometric matrix which consists of four subsystems as defined in Figure 2.4. The pss
metabolites are denoted as xpss, the corresponding rates as vpss. The subsystem Speq is constructed by
removing all non-pss metabolites and non-pss reactions from the stoichiometric matrix.
vpss
vdyn
xpss
Speq
S2
xdyn
S3
S4
Figure 2.4: Arrangement stoichiometric matrix S
From equation (2.85) it follows that the reaction rate of pseudo steady state metabolites, denoted as vpss,
lies in the nullspace of Spps. The number of independent pss rates can be determined from the rank analysis
of Spss and these determine the rates of all pss rates. This dependence can be found by determining the
rational basis of the null space of Spss, denoted as Lpss.
Subsequently the matrix Svpss is created which consist of the submatrixes of Spss & S3 which is multiplied
by Lpss.
Slump = S vpss ⋅ L pss
(2.92)
Slump contains the lumped stoichiometries of the reactions and is used to replace the submatrix Svpss in S.
This results that the rows of the pss metabolites only contains zeros and can be removed which reduces the
stoichiometric matrix.
The theory will be illustrated by the following example
Example 2.2
Let’s consider the following reaction scheme
30
v1
v2
A
X
v3
B
XH
X
C
v4
XH
Reaction Scheme 4
Here B is assumed to a pss metabolite so that v2 is equal to v3. This reaction scheme is actually a
simplification of the oxidative branch of the pentose phosphate route in which metabolite B is represented
by 6Pgluct and X and XH are the cofactors NADP and NADPH respectively. The stoichiometric matrix of
this reaction scheme is:
S full
⎡ 1 −1 0 0 ⎤
⎡ 1 −1
⎢ −1 0 1 0 ⎥
⎢ −1 0
⎢
⎥
elimination
⎢
S
= ⎢ 0 1 0 −1⎥ ⎯⎯⎯⎯
⎯
→
=
dependencies
⎢0 1
⎢
⎥
1
1
0
0
−
−
⎢
⎢
⎥
⎣ −1 − 1
⎢⎣ 1 1 0 0 ⎥⎦
0⎤
0 ⎥⎥
0 −1⎥
⎥
0 0⎦
0
1
The stoichiometric matrix is organized as [B A C X XH] in the rows and as [v2 v3 v1 v4] in the columns.
In this way the pss metabolite B and the involved reactions are the left corner of the stoichiometric matrix.
Rank analysis shows that the the 5th and 6th row are dependent and hence, a bit arbritary, the 6th rows
which represents the cofactor XH is omitted. From the resulting independent matrix S the non-pss
metabolites A, C and X and the non-pss reactions v1 and v4 are removed to construct Spss. Subsequently
the null space on a rational basis is calculated and the results are shown below.
⎡1⎤
S pss = [1 −1] ⇒ L pss = ⎢ ⎥
⎣1⎦
The submatrix Svpps consists of the first two columns of S and is multiplied with the nullspace Lpss which
results in:
Slump
⎡0⎤
⎢ −1⎥
=⎢ ⎥
⎢1⎥
⎢ ⎥
⎣ −2 ⎦
This matrix shows that one A and 2 X are converted into one C and 2 XH (remember the dependency
between X and XH). Subsequently Slump is used to replace Svpss in matrix S. The resulting reduced
stoichiometric matrix is:
S red
⎡0
⎢ −1
=⎢
⎢0
⎢
⎣ −2
0 0⎤
⎡ −1 1 0 ⎤
1 0 ⎥⎥
∼ S red = ⎢⎢ 0 0 0 ⎥⎥
0 1⎥
⎢⎣ −2 0 0 ⎥⎦
⎥
0 0⎦
31
Here the columns are are arranged as [v3, v1 and v2], the row arrangedment did not change. A bit arbritary
v3 is chosen to act as an independent rate which determined the rate of v2. All entries with respect to the
pss metabolite B are zero, i.e. the first row, and as a consequence this row can be omitted from the matrix.
32
Experimental Part
During this project no experiments have been performed and hence this chapter only discusses the used
software packages.
2.7 Computational tools
The development of reduced stoichiometric models for Penicillium chrysogenum and subsequently
metabolic network analysis and data reconciliation has been conducted using the software In Silico
(v1.2.4, 2006). In Silico is an integated, platform independent computational tool for graphically oriented
reconstruction, management and engineering of large-scale cellular network (In Silico manual).
In Silico has the advantage that it visualizes the metabolic network, by identifying parallel routes, dead
ends, and conserved moieties. In addition it can identify which degrees of freedom have to be set in order
to get a closed system.
Furthermore, unlike Matlab, it does not require balanced net conversion rates as an input, as the measured
rates are automatically balanced by the stoichiometric of the entered metabolic network reactions. This is
the main reason why In Silico has been used to calculate the metabolic fluxes.
Although metabolic network analysis has also been performed by Matlab (v2006b), it requires much more
mathematical actions as:
•
A rank analysis has to be performed so that the dependencies can be removed.
•
Measured net conversion rates need to be balanced first
•
Checking of possible dead ends and parallel routes
The metabolic fluxes are calculated both with Matlab and In Silico and showed no difference with the
provided metabolic fluxes of van Gulik et al. (2000).
Matlab is mainly used for simulating the dynamic behaviour of the metabolic system and to balance the
net conversion rates. The calculation unmeasured metabolites has been conducted with Excel (Office
2003).
The Matlab files and the In Silico database file can be found in appendix F.
33
3 Results
3.1 Calculation of net conversion rates
The unbalanced conversion rates can be calculated by setting up a mass balance over the reactor which
will be based on the experimental data listed in appendix A. The rearranged mass balances are:
qglc = ( Duit ⋅ C glc − Din ⋅ C glc , feed ) ⋅ M w C x
(4.1)
q paa = ( Duit ⋅ C paa − Din ⋅ C paa , feed ) ⋅ M w C x
qO 2 =
⎞
Fgas ⋅ M w ⋅ 10 ⎛ (100 − X O 2, g , feed − X CO 2, g , feed )
⋅⎜
⋅ X O 2, g − X O 2, g , feed ⎟
⎜
⎟
V ⋅ Cx
(100 − X O 2,g − X CO 2,g )
⎝
⎠
qCO 2 =
⎞
Fgas ⋅ M w ⋅ 10 ⎛ (100 − X O 2, g , feed − X CO 2, g , feed )
⋅⎜
⋅ X CO 2, g − X CO 2, g , feed ⎟
⎜
⎟
V ⋅ Cx
(100 − X O 2,g − X CO 2,g )
⎝
⎠
(4.2)
(4.3)
μ = Duit ⋅ 1000
(4.4)
q penG = Duit ⋅ X penG ⋅ M w C x
(4.5)
qtoc = Duit ⋅ ( X toc − 6 ⋅ X glc − 16 ⋅ X penG − 8 ⋅ X paa ) ⋅ M w C x
(4.6)
Where qglc, qpaa, qO2, qCO2, m, qpenG and qtoc are the biospecific net conversion rates of glc, PAA, O2, CO2,
biomass, penG and byproduct respectively which are expressed in mmol/(Cmol*hr). The numbers 6, 16
and 8 in equation (4.6) are the number of carbons in glucose, penicillin G and PAA respectively. The byproduct contains 36% ExPept and 64% psacch and it will be assumed that this ratio remains constant
under dynamic conditions.
The result of the calculation is shown in Table 3.1 in which the unbalanced net conversion rate is
presented in mmol/Cmol/hr and in μmol/gDW/s. The standard deviation is denoted as σqi, and is together
with the corresponding net conversion rate, entered in In Silico in order to calculate the steady state
metabolic fluxes. The standard deviation can be determined from the the variance/covariance matrix, PRm,
which can be calculated by:
PRm = R jac ⋅ B ⋅ R jacT
(4.7)
with
Rjac = jacobian(rm,v)
B = diag(var)
(4.8)
(4.9)
34
Table 3.1: Unbalanced net conversion rates
qi
σqi
μmol/gDW/s
mmol/Cmol/hr
glucose
σqi
qi
-19.217
0.718
-0.19037
0.00712
-0.421
0.067
-0.00417
0.00067
-56.512
11.528
-0.55984
0.11432
CO2
51.469
3.874
0.50987
0.03842
biomass
49.761
1.113
0.49295
0.01102
0.327
0.048
0.00324
0.00048
byprod
12.046
1.505
0.11933
0.01493
ExPept
4.298
0.537
0.04257
0.00533
psacch
7.748
0.968
0.07676
0.00960
PAA
O2
PenG
The covariance/variance matrix is shown in Table 3.2. The standard deviation can be calculated by taking
the square root of the variances which are one the diagonial of Prm.
Table 3.2: Covariance/Variance Matrix Prm
cov
glc
PAA
O2
CO2
biomass
Pen-G
byprod
glc
5.15E-01
8.04E-03
1.08E+00 -9.84E-01
-3.82E-01 -6.25E-03 -2.30E-01
PAA
8.04E-03
4.54E-03
2.36E-02 -2.15E-02
-4.90E-03 -1.14E-04 -3.47E-02
O2
1.08E+00
2.36E-02
1.33E+02 -5.29E-01 -1.12E+00 -1.84E-02 -6.77E-01
CO2
-9.84E-01 -2.15E-02
-5.29E-01
1.50E+01
1.02E+00
1.67E-02
6.17E-01
biomass
-3.82E-01 -4.90E-03 -1.12E+00
1.02E+00
1.24E+00
8.13E-03
3.00E-01
PenG
-6.25E-03 -1.14E-04
-1.84E-02
1.67E-02
8.13E-03
2.33E-03 -3.10E-02
byprod
-2.30E-01 -3.47E-02
-6.77E-01
6.17E-01
3.00E-01 -3.10E-02
2.27E+00
3.1.1 Balancing net conversion rates
The net conversion rates are balanced according to the procedure described by van der Heijden (van der
Heijden et al. 1994).
The elementary composition matrixes are shown in Table 3.3 and Table 3.4. Since sulphate and phosphate
are not measured, the elements S and P will be excluded. The biomass composition is based on the
composition given by van Gulik as mentioned in chapter 2.1.
35
Table 3.3: Elementary composition matrix (Em)
glc PAA O2 CO2 biomass penicillin byprod
C
6
8
0
1
1.000
16
1.0000
H
12
8
0
0
1.794
18
1.6393
N
0
0
0
0
0.162
2
0.1035
O
6
2
2
2
0.578
4
0.6395
+/-
0
0
0
0
-0.012
0
0.0000
Table 3.4: Elementary composition matrix (Ec)
NH4 H2O H
C
0
0
0
H
4
2
1
N
1
0
0
O
0
1
0
+/1
0
1
The net conversion rates can be balanced by employing the following constraint which follows from the
elementary balances:
E⋅r = 0
(4.10)
where E is the elementary composition matrix and r is the vector consisting of all observable and nonobservable conversion rates. This equation can be rewritten into to:
Em ⋅ rm + Ec ⋅ rc = 0
(4.11)
where rm denotes the vector of all measured conversion rates, rc the non-measured part of the conversion
rates and Em and Ec the elementary composition matrixes that correspond to rm and rc respectively.
Now that all the necessary data is collected, the conversion rates can be balanced and if necessary be
calculated. Dekok and Roels proposed a relative easy and fast method for balancing conversion rates
(Dekok and Roels 1980). Briefly the equations of their method are:
{
rc = − EcT ⋅ ( Em ⋅ Prm ⋅ EmT ) ⋅ Ec
−1
}
−1
(
⋅ Ec T ⋅ ( Em ⋅ Prm ⋅ EmT ) ⋅ Em ⋅ rm
(4.12)
)
(4.13)
−1
rm = − Prm ⋅ EmT ⋅ ( Em ⋅ Prm ⋅ EmT ) ⋅ Em ⋅ rm + Ec ⋅ rc + rm
−1
The balanced vectors are denoted with a ^. However as already mentioned the rates will be balanced
according to the procedure of van der Heijden that employs the redundancy matrix R which provides
insight whether a system is balanceable and what rates are observable.
The vector rc can be calculated by rewriting equation (4.11):
rc = −Ec −1 ⋅ Em ⋅ rm
(4.14)
36
However Ec is a non-square matrix (5x3) and therefore its pseudo-inverse, denoted as Ec#, needs to be
calculated. If the matrix EcTEc is not singular the pseudo-inverse can be calculated according to:
E c # = ( E c T ⋅ Ec ) i E c T
−1
The pseudo-inverse in this case is calculated the same as the normal inverse. Singular value
decomposition is used when the system is singular. However due to the fact that Ec is not square, the
singular value decomposition of EcTEc will be calculated, i.e. in short:
[U S V] = svd (EcTEc)
(4.15)
The pseudo-inverse of this matrix is equal:
(E
T
c
⋅ Ec ) = V ⋅ S # ⋅ U T
(4.16)
Since S is a diagonal matrix, S# can be calculated according to:
S
S
#
= diag (a1, a2, …, an)
(4.17)
= diag (α1, α2, …, αn)
(4.18)
If ai is unequal to zero αi=1/ai, else αi=0. The redundancy matrix can be calculated according to:
R = Em − Ec ⋅ Ec # ⋅ Em
(4.19)
If the redundancy matrix is multiplied by a vector containing the true conversion rates, the result will be a
zero vector.
R ⋅ rm = 0
(4.20)
A system is balanceable if and only if there is a redundancy in measurements. The redundancy in
measurements is the number of independent equations in equation (4.20) and equals the rank of matrix R.
In this case the rank of the redundancy matrix is 2, which shows this system is balanceable. Furthermore
zero columns in matrix R show that although the corresponding rate is measured, it can not be balanced
since it will not be used in equation (4.20). The redundancy matrix is shown in Table 3.5.
Table 3.5: Redundancy matrix
R glucose PAA
O2
CO2
biomass
PenG
byprod
C
6
8
0
1
1
16
1
H
0
0.27
-0.27
-0.27
0.01
0.27
0
N
0
-0.8
0.8
0.8
-0.03
-0.8
-0.01
O
0
-0.53
0.53
0.53
-0.02
-0.53
-0.01
-/+
0
-0.27
0.27
0.27
-0.01
-0.27
0
The estimated (balanced) conversion rate vectors rm and rc can be calculated according to:
37
rm = ( I − Prm iR T iPε −1 iR )irm
(4.21)
rc = −E#c ⋅ Em ⋅ rm
(4.22)
with
Pε = R ⋅ Prm ⋅ R T
(4.23)
The test function, denoted as h, is defined as:
h = εT ⋅ Pε−1 ⋅ ε
(4.24)
with
ε = R ind ⋅ rm
(4.25)
Here Rind is the independent redundancy matrix. According to van der Heijden the reduction of R to Rind
will always result in the existence of the inverse of Pe. In this test function the residuals, denoted as ε are
weighted according to their accuracy. Van der Heijden showed that that h possesses a chi-square
distribution regardless of any correlations in the residuals. The rank of Pe, is equal to the degree of
freedom of the chi-square distribution.
The calculated balanced rates, rm_est can be found in Table 3.6. The confidence level for measurement
errors or a wrong system definition is 8%. Balancing with the program In Silico resulted in the same
values for the net conversion rates.
Table 3.6: Balanced conversion rates
rm_est
mmol/Cmol/hr
glucose
PAA
-19.289 PenG
-0.327 byprod
rm_est
mmol/Cmol/hr
0.327
12.038
-48.047 NH4+
-10.033
CO2
51.275 H2O
75.278
biomass
49.740 H+
O2
8.075
3.1.2 Adjusting biomass definition
Uly Nasution has shown that in energy limited environment the biosynthesis of storage materials is greatly
enhanced (Nasution et al. 2006). To focus on this behaviour the biomass formation is separated into a
synthetic (syn_X, l18.2) and a storage trehalose part (tre_X, l17.6). Note the mentioned reactions can be
38
found in appendix B. The steady state metabolic flux of the biomass made up by storage material (tre_X)
is equal to:
qtre _ X ,new =
vtre , X ⋅ μold
(4.26)
1 − vtre, X
in which vtre,X is the stoichiometric coefficient of t6P in the orginal biomass synthesis reaction (l18.1), μold
the biomass growth rate based on the old definition and qtre_X,new the biomass specific storage material part
of the biomass synthesis. The stoichiometric coefficient vtre,X is equal to 0.00107 on the basis of 1 Cmol X.
Since the biomass definition has been changed and solely consists of the synthetic part now, all biomass
specific net conversion rates for a certain compound i need to be adjusted according to:
qi ,old
qi ,new =
(4.27)
1 − vtre, X
in which qi,old is the specific biomass net conversion rate of i based on the old biomass definition and qi,new
for the new biomass definition. The growth rate for the synthetic part of the biomass remains the same, i.e.
μnew =
(1 − v ) ⋅ μ
tre , X
1 − vtre, X
old
= μold = μ
(4.28)
The measured net conversion rates of the new definition are shown in Table 3.7. The measured net
conversion rates defined on the basis of the synthetic biomass composition have been entered in In Silico.
The corresponding standard deviation, σi,new has been calculated according to:
σ i.new =
σ i ,old
qi ,old
.qi ,new
(4.29)
Table 3.7: Unbalanced net conversion rates based on new biomass definition
qi
qi
σqi
σqi
μmol/gDW/s
mmol/Cmol/hr
glucose
-19.237
0.718
0.191
0.00712
-0.421
0.067
0.004
0.00067
-56.573
11.540
0.560
0.11432
CO2
51.524
3.878
0.510
0.03842
biomass
49.761
1.113
0.493
0.01102
0.327
0.0483
0.003
0.00048
12.059
1.507
0.119
0.01493
ExPept
0.053 0.00119
0.001
0.00001
psacch
4.302
0.043
0.00533
PAA
O2
PenG
byprod
0.538
39
3.2 Model Reductions
In this chapter the results of the various model reductions as elaborated in chapter 2.6 are presented.
3.2.1 Data driven reduction of stoichiometric model
Due to the limited number of the measurements compared to the number of kinetic parameters, the
metabolic reaction network is reduced step by step. For each major reduction, the new stoichiometric
model has been implemented in In Silico, see Table 3.8, to see the effect on the steady state metabolic
fluxes. The table contains information about the number of compartments, metabolites (intracellulars
within the parenthesis), number of reactions, data used and the chi square of the In Silico model.
Table 3.8: In Silico models developed
model
compartments
penG
cytosol (top), mitochondria
metabolites
reactions
data
X2
188 (169)
167
(van Gulik et al. 2000)
100%
& peroxisome
penGv2
cytosol (top), mitochondria
163 (155)
145
(Nasution et al. 2006)
92.1%
penGv3
cytosol (top), mitochondria
112 (98)
94
(Nasution et al. 2006)
92.1%
penGv4
cytosol (top)
89
75
(Nasution et al. 2006)
92.1%
3.2.1.1 Original stoichiometric model (penG)
The original stoichiometric model for Penicillium chrysogenum, denoted as penG, contains 169
intracellular metabolites, 18 extracellular metabolites and 167 metabolic reactions2 (van Gulik et al. 2000).
The model contains three compartments (cytosol, mitochondria and peroxisome). The number of degrees
of freedom of this model is 11.
Rank analysis shows that the stoichiometric matrix of the intracellular metabolites has a rank of 167 and is
rank deficient in the rows. The row dependencies are resulting by the conserved moieties of this system,
which are
1 NADH:cyt + 1 NAD:cyt = ∑1
(4.30)
2
The orginal model of van Gulik actually contains 166 reactions. However this stoichiometric model has one dead
end, which is caused by the PIO synthesis, which is produced but not transported across the plasma membrane.
Hence the transport step r9.23 has been added so that the resulting metabolic network has no dead ends.
40
1 NADPH:cyt + 1 NADP:cyt = ∑2
(4.31)
1 NADH:mit + 1 NAD:mit = ∑3
(4.32)
1 AcCoA:mit + 1 HCoA:mit + 1 succCoA:mit = ∑4
(4.33)
1 NADPH:mit + 1 NADP:mit = ∑5
(4.34)
1 ATP:mit + 1 ADP:mit = ∑6
(4.35)
1 FADH2:mit + 1 FAD:mit = ∑7
(4.36)
1 ADP:per + 1 ATP:per = ∑8
(4.37)
1 H:per + 1 Pi:per = ∑9
(4.38)
1 PAACoA:per + 1 HCoA:per = ∑10
(4.39)
1 HCoA:cyt + 1 AcCoA:cyt + 1 steaCoA:cyt + 1 olCoA:cyt + 1 linCoA:cyt = ∑11
(4.40)
1 MYTHF:cyt + 1 THF:cyt + 1 FTHF:cyt + 1 METHF:cyt = ∑12
(4.41)
1 ADP:cyt + 1 ATP:cyt + 1 SAH:cyt + 1 SAM:cyt + 1 A:cyt + 1 PAPS:cyt = ∑13
(4.42)
Note that AMP is not considered to be a conserved moiety. A closer examination of the network shows
that AMP is being synthesized from IMP (r13.2) and being converted into RNA (r14.1) and is not
involved in any other reactions which are the result that AMP has largely been lumped with the adenylate
kinase reaction. Hence its concentration profile is dependent on the conversion rates of these reactions and
not any conservation relation.
This stoichiometric model has been implemented in the In Silico software in order to reproduce the steady
state fluxes as calculated by van Gulik (van Gulik et al. 2000). The resulting metabolic fluxes are listed in
appendix B and as can be seen from Table B. 1 is the results are similar.
The stoichiometric model contains many metabolites that have not been measured during the glucose
response experiment or at steady state and hence a new data-driven stoichiometric model is developed,
which will be elaborated in the next chapters.
3.2.1.2 Stoichiometric model penGv2
The stoichiometric model is adjusted for the experimental data provided by Uly Nasution, Table 3.8, and
will be denoted by PenGv2. In this model the relatively small peroxisome compartment is omitted, which
includes the biosynthesis of penicillin, and several related by-products. The by-products with respect to
the penicillin biosynthesis, e.g. 6APA, OPC, 8HPA, are neglected since these have not been measured and
are minor carbon sinks. Only the total by-product conversion rate that includes psacch, ExPept and the
penicillin related by-products are measured. The metabolic fluxes of all penicillin related by-products is
41
7% of the total by-product rate which has a standard deviation of 12%. As a consequence the error made
by omitting the by-products falls within the large standard deviation of the total by-product rate
The stoichiometric model has been reduced to 150 intracellular metabolites, 13 external metabolites, and
145 reactions and contains two now compartments, the cytosol and the mitochondria. The number of
conserved moieties is 10 since the peroxisome related conserved moieties do not hold anymore. Omitting
the penG biosynthesis by-products resulted that the degrees of freedom of the system has significantly
reduced to 5.
3.2.1.3 Stoichiometric model penGv3
Since only a limited amount of metabolites has been measured, many undeterminable compounds, e.g
RNA, chor, bIM, ery etc, have been elimated by lumping the involved reactions, see Table 3.9. The model
has been largely reduced by elimating metabolites from the secondary pathways such as the amino acid
synthesis (r11.i), nucleotide synthesis (r13.i), fatty acid synthesis (r16.i) and the biosynthesis of glycogen
and polysaccharides. Also a few metabolites from the glycolysis have been elimated such as 2PG and
13PG since these are relative unimportant, i.e. they are not involved in a dynamic reaction nor are they
effectors of them. Glucose has been elimated due to its very low concentration in the cell. Although the
penicillin biosynthesis is of major interest, the corresponding intermediates have not been measured and
hence the reaction has been lumped.
The calculated metabolic fluxes from this new In Silico model, penGv3, correspond to those of PenGv2
and are shown in appendix B.
In addition the separation of the synthetic and the trehalose part of the biomass, as discussed in chapter
3.1.2 has been incorporated in this stoichiometric model, which requires the input of the net conversion
rates listed in Table 3.7. This requires the input of one addition transport step for tre, which increases the
degree of freedom of the system to 6.
The number of intracellular and extracellular metabolites has been reduced to 98 and 112 respectively and
the network holds 94 reactions. The number of conserved moieties remains the same since the
mitochondrial compartment has not been discarded yet, which shall be done in the following
stoichiometric model.
42
Table 3.9: Lumping scheme penGv3
notation biosynthesis of lumped reactions
eliminated metabolites
l1.1
g6p
r1.1, r9.3
glc
l1.2
3PG
r1.5, r1.6
13PG
l1.3
PEP
r1.7, r1.8
2PG
l11.1
val
r11.23, r11.24
aKI
l11.2
leu
r11.23, r11.25, r10.19, r11.26
aKI, bIM
l11.3
trp
r11.27, r11.30, r11.31
chor, PRPP
l11.4
tyr
r11.27, r11.29
chor
l11.5
phe
r11.27, r11.28
chor
l11.6
arg
r11.4, r11.5, r11.6, r10.7
carbP, ctl
l11.7
cys
r11.11, r11.12, r11.15, r7.2
Ac, PAPS, H2S
l11.8
homcys
r11.11-r11.14, r7.2, r11.18
Ac, PAPS, H2S, AcHomser
l11.9
thr
r11.18, 11.19
homser
l11.10
his
r11.31, r11.32
PRPP
l13.2
GMP
r11.31, r8.3, r13.1, r13.3
PRPP, FTHF, IMP
l13.3
AMP
r11.31, r13.1,r8.3, r13.2
PRPP, FTHF, IMP
l13.4
UTP
r11.31, r13.4, 13.5
PRPP, UMP
l17.1
t6P
r13.9, r17.5, r17.6
UDP, UDPglc
l17.3
g1P
l17.4
psacch
l17.7
tre:ext
l18.2
biomass
introducing g1P
r17.1
g6P replaced by g1P in this reaction
introducing tre transport step
r11.33, r12.1, r12.2, r13.7,
AApool, Aaprotsyn, PROT, CMP, RNA,
r14.1, r8.3, r8.4, r13.8, r16.1-
FTHF, SAM, A, ino, gcl3P, steaCoA,
r16.16, r17.2-r17.4, r17.8,
olCoA, linCoA, phospht, CDPDAcgcl,
mu0.05.
CMP, pHeta, Phchol, Phino, Phser,
TRIA, meva, lano, ergo, ESE, chit, m1P,
man, ery
l19.1
penG
r19.1-r19.4
ACV, iPN, PAACoA
43
3.2.1.4 Stoichiometric model penGv4
However only the average cell concentration has been measured and the individual concentrations within
the cytosol and the mitochondria are still unknown. Moreover no concentration ratios between the
compartments for Penicillium chrysogenum have been known. In Table 3.10 the metabolites that are
present in both the cytosol and the mitochondria are listed.
Table 3.10: Metabolites that are present in the cytosol and mitochondria
Glycolysis:
pyr
TCA metabolites:
citr, iCitr, mal, OAA, fum
Amino Acid Synthesis:
glu, ile, val, aKG, thr
Cofactors:
ATP, ADP, NADH, NAD, NADPH, NADP, AcCoA, HCoA
Miscellanous:
Pi, H, H2O, CO2, O2,
Due to the absence of data with respect to the compartmentation, the mitochondrion has been omitted so
that the model is reduced to a one compartment model. As a consequence of omitting the mitochondria
compartment, the developed stoichiometric model now contains parallel routes. These need to be
eliminated to avoid that any inner degrees of freedom need to be set. The metabolic network contains 5
parallel routes:
1. r4.5 (mitochondria) with r4.6 (cytosol)
2. r4.11 (mitochondria) with r5.3 (cytosol)
3. r4.1 (mitochondria) + r5.2 (cytosol) with ATP hydrolysis (cytosol)
4. glycolysis + TCA cycle + PPP (cytosol) + l6.1 + l6.2 + ATP hydrolysis (cytosol)
5. l6.2 and l6.3
The first two routes are parallel because apart from the location the reactions are identical and hence any
one of them can be discarded. The anaplerotic reaction r5.3, OAA is converted into mal, proceeds in the
opposite direction as r4.11. Studying the metabolic fluxes shows the metabolic flux of r5.3 is 25% of that
of r4.11. Furthermore it seems that the cytosolic malate flux is used to drive the transport step r10.14,
which transports mitochondrial citrate across the mitochondrial membrane while moving malate the
opposite way. Since this transport step has been omitted, there is no need for r5.3 anymore and hence it is
discarded. In the anaplerotic reaction r5.2, citr is used to produce AcCoA and has a metabolic flux that is
19% of that with r4.1. Hence it will be omitted from the stoichiometric model to keep the TCA cycle
intact.
44
The fourth parallel route consists of many reactions and it’s not so straightforward to see what is causing
it. In Silico contains an observability test that determines which rates need to be specified in order to close
the system. It revealed that there are alternative pathways for the biosynthesis of NADPH which are
located in the oxidative branch of the PPP and the isocitrate dehydrogenase reaction.
The main biosynthesis of NADPH is catalyzed by the enzymes in the oxidative PPP, i.e. 75% of the total
NADPH flux, and hence the isocitrate dehydrogenase has been omitted.
The fifth parallel route is caused by the oxidative phosporylation of NADH:mit and NADH:cyt which can
be eliminated by lumping them based on their steady state flux distribution. This is discussed in chapter
3.2.2.
With respect to the previous model penGv3, the biosynthesis of arg (l11.6) has been unlumped into l11.11
and l11.12, due to the fact that the intermediate ornithine has been measured.
The stoichiometric model still contains 6 degrees of freedom. The number of conserved moieties of this
system has been reduced due to the omitting of the mitochondria:
[ ATP ] + [ ADP ] = ∑ ATP
(4.43)
[ NADH ] + [ NAD ] = ∑ NAD
(4.44)
[ NADPH ] + [ NADP ] = ∑ NAD
(4.45)
[ FADH 2] + [ FAD ] = ∑ FAD
(4.46)
[ HCoA] + [ AcCoA] + [ succCoA] = ∑CoA
(4.47)
[THF ] + [ MYTHF ] + [ METHF ] = ∑THF
(4.48)
The metabolic network of Penicillium chrysogenum is schematically presented in Figure 3.1. The
concentration of metabolites that are written in italic can be calculated since these are involved in
reactions that can be considered as near equilibrium, see chapter 2.6.2. However those which have been
underlined assumptions are needed. The remaining metabolites have been measured.
The resulting metabolic reaction network contains 75 intracellular components, 14 externals compounds
and 75 reactions. In addition the top compartment is now the fermentor compartment and as a
consequence 14 additional transport reactions are added in the In Silico model which represents the
biospecific net conversion rates as calculated in chapter 3.1. The characteristics of the current metabolic
network can be found in Table 3.11.
45
External
glc
Cytosol
tre_X
vl1.1
vl17.6
tre
vr17.7
t6P
vl17.1
g6P
psacch
ExPept
vr17.4
vr2.1
6Pgluct
vr2.2
vr11.34
f6P
ADP
Xylu5P
Rib5P
GAP
sed7P
E4P
f6P
vl1.3
AMP
f16P
vr15.1
ADP
FADH2
vl6.1
FAD
NADH
vl6.4
NAD
vr8.1
CO2
CO2
O2
O2
vr9.2
his
UTP
vr13.6
CTP
GAP
cys
vl11.7
3PG
THF
vl11.10
vl13.1
vl13.3
ATP
GMP
vl13.2
ATP
NH4
Ribu5P
g1P
METHF
phe
vl11.5
tyr
vl11.4
trp
vl11.3
NH4
vr11.9
ser
-
gly
PEP
vr11.22
ala
pyr
vl11.1
val
vr4.1
vl11.2
leu
vr1.9
vl19.1
PenG
PAA
vr5.1
SO4
vr9.4
SO4
ile
met
Pi
vr9.2
vr11.21
vr11.20
AcCoA
thr
vl11.9
asn
vr11.17
homcys
vl11.8
asp
vr11.16
OAA
citr
mal
iCitr
vr4.4
Pi
Conserved moieties:
AcCoA + HCoA + succCoA = ∑CoA
ATP + ADP + AMP = ∑ATP
NADPH + NADP = ∑NADP
NADH + NAD = ∑NAD
THF + METHF + MYTHF = ∑THF
FADH2 + FAD = ∑FAD
vl18.2
fum
aKG
vr4.7
succ
glu
vr11.7
aAd
vr11.3
pro
vl11.11
orn
vr11.2
gln
vr11.8
lys
vl11.12
arg
syn_X
succCoA
Figure 3.1: Schematic metabolic network for Penicillium chrysogenum
Table 3.11: Characteristics metabolic network penGv4
Number of
total reactions
75
Dynamic reactions see chapter 3.3.
52
PEQ relations see chapter 3.2.4.
23
intracellular metabolites
73
extracellular metabolites
14
Degree of freedom
6
Entered measured net conversion rates
7
Chi square
93%
Parallel routes
none
46
However the mitochondrion can be considered as the cell’s power plant, since it is the major site of
oxidative metabolism, such as the TCA cycle and the oxidative phosphorylation. Omitting the
mitochondria and averaging the concentration listed in Table 3.10 may lead that the energy housekeeping
of the cell may be wrongly described. For example, the highly exergonic oxidation of mitochondrial
NADH produces much more ATP (2.5 ATP/NADH:mit) than cytosolic NADH (1.5 ATP/NADH:cyt).
Averaging the NADH would describe the amount of ATP produced in the cell incorrectly. Note that the
mentioned ATP/NADH is theoretical and based on the stoichiometric model (r6.1, r6.2 and r6.4).
Comparison of the calculated metabolic fluxes between penGv3 and the penGv4 model confirms the fact
that omitting the mitochondria and subsequently the removal of the parallel routes will lead to incorrect
prediction of the cell’s haviour, see Table 3.12.
The increase in metabolic fluxes in the PPP, is caused by the omital of the isocitrate dehydrogenase
(NADP) reaction which forces the cell to enhance its flux through the oxidative branch of PPP in order to
satisfy the NADPH requirement. This results that more carbon is withdraw from the glycolysis in favour
of the PPP which also results that the flux through the TCA cycle is decreased. However this effect is less
profound due to the fact that the non-oxidative part of the PPP supplies some of the withdrawn carbon
back to the glycolysis.
As can be seen especially the fluxes with respect to the oxidative phosphorylation are significant. The
oxidation of FADH2 (l6.8) has decreased due to the decreased flux in the C4 pool in the TCA cycle
whereas the oxidation of the NADH has increased due to the significant increase of NADH production by
isocitrate dehydrogenase (NAD) reaction, which again is caused due to the fact that isocitrate
dehydrogenase (NADP) has been omitted.
Hence omitting the isocitrate dehydrogenase (NADP) reaction (r4.5, r4.6) will lead to significant chances
in the metabolic fluxes of the oxidative phosphorylation and pentose phosphate route. The alternative is to
include this reaction in the reaction network but this has the disadvantage that one inner degree of freedom
has to be set.
47
Table 3.12: Comparison fluxes penGv3 & penGv4
penGv4
penGv3
change
l6.8
0.8208 l6.1+l6.3
0.4386
87.1%
l6.7
0.1170 l6.2
0.2398
51.2%
r4.4
0.1329 r4.4
0.0938
41.7%
r2.7
0.0268 r2.7
0.0190
41.2%
r2.4
0.0584 r2.4
0.0427
36.6%
r2.1
0.0944 r2.1
0.0709
33.1%
r2.2
0.0944 r2.2
0.0709
33.1%
r2.6
0.0316 r2.6
0.0237
33.0%
r2.5
0.0316 r2.5
0.0237
33.0%
r4.11
0.1228 r4.11
0.1753
29.9%
r1.2
0.0607 r1.2
0.0842
27.9%
r2.3
0.0360 r2.3
0.0282
27.7%
r4.2
0.1329 r4.2
0.1753
24.2%
r15.1
1.1664 r15.1
0.9618
21.3%
r1.4
0.1138 r1.4
0.1216
6.4%
r1.3
0.1138 r1.3
0.1216
6.4%
r4.9
0.1170 r4.9
0.1248
6.3%
r4.8
0.1170 r4.8
0.1248
6.3%
r4.7
0.1170 r4.7
0.1248
6.3%
r4.10
0.1228 r4.10
0.1306
6.0%
r4.3
0.1329 r4.3
0.1407
5.6%
r4.1
0.1714 r4.1
0.1793
4.4%
r1.9
0.2314 r1.9
0.2392
3.3%
l1.3
0.2397 l1.3
0.2475
3.2%
l1.2
0.2539 l1.2
0.2617
3.0%
R9.1
0.3868 R9.1
0.3959
2.3%
3.2.2 Lumping and adjusting the oxidative phosphorylation
Since the mitochondria compartment has been omitted the oxidative phosphrylation reaction need to be
adjusted. Until now the theoretical P/O ratio for the oxidation of mitochondrial NADH that has been used
48
is 2.5 which can be calculated from reaction r6.1 and r6.4. However van Gulik et al. estimated the P/O
ratio for the oxidation of mitochondrial NADH of 1.84 (vanGulik et al. 2001). As such the stoichiometric
coefficients of mitochondrial and cytosolic protons need to be adjusted by multiplying them with the ratio
of the estimated P/O ratio to the maximum P/O ratio. Since one proton is associated with NADH, the
stoichiometric coefficient of mitochondrial H needs to be subtracted by one before it is multiplied. The
result is shown below:
8.36 H:mit + NADH:mit + 0.5 O2 => 7.36 H:cyt + H2O:cyt + NAD:mit
(l6.1)
5.416 H:mit + NADH:cyt + 0.5 O2 => 4.416 H:cyt + H2O:cyt + NAD:mit
(l6.2)
4.416 H:mit + FADH2:mit + 0.5 O2 => 4.416 H:cyt + H2O:cyt + FAD:mit
(l6.3)
Subsequently the formation of ATP which is driven by the inward flux of cytosolic protons into the
mitochondrial (r6.4) is lumped with the phosphate & water transport (r10.5 and r10.3 respectively) and the
ADP/ATP shuttle (r10.1) in order to eliminate all mitochondrial reactants with exception of H:mit.
ADP:mit + 4 H:cyt + Pi:mit
=> ATP:mit + 3 H:mit + H2O:mit
(r6.4)
ADP:cyt + ATP:mit
=> ADP:mit + ATP:cyt
(r10.1)
H2O:mit
=> H2O:cyt
(r10.3)
H:cyt + Pi:cyt
=> H:mit + Pi:mit
(r10.5)
ADP:cyt + 5 H:cyt + Pi:cyt
=> ATP:cyt + 4 H:mit + H2O:cyt
(l6.4)
The only mitochondrial compound in this reaction is H:mit. Implementing reactions l6.1-3 in the In Silico
model has shown that 90% of the production of H:mit is caused by l6.4 and that l6.1-l6.3 are the major
sinks for it (99%). Therefore H:mit in the reactions r6.1-3 will be eliminated by l6.4. This yields the
following lumped reactions:
2.09 ADP:cyt + 3.09 H:cyt + NADH:mit + 0.5 O2:cyt + 2.09 Pi:cyt => 2.09 ATP:cyt + 3.09 H2O:cyt +
NAD:cyt
(l6.5)
1.354 ADP:cyt + 2.354 H:cyt + NADH:cyt + 0.5 O2:cyt + 1.354 Pi:cyt => 1.354 ATP:cyt + 2.354
H2O:cyt + NAD:cyt
(l6.6)
1.104 ADP:cyt + 1.104 H:cyt + FADH2:mit + 0.5 O2:cyt + 1.104 Pi:cyt => 1.104 ATP:cyt + 2.104
H2O:cyt + FAD:cyt
(l6.7)
49
Since the model does not contain any inner compartments, reactions l6.5 and l6.6 are parallel to each
other, and are therefore lumped based on their flux distribution (70.5 to 29.5% respectively). This yields
the following lumped reaction:
1.87 ADP + 2.87 H + NADH + 0.5 O2 + 1.87 Pi => 1.87 ATP + 2.87 H2O + NAD
(l6.8)
3.2.3 Calculation of unmeasured metabolites
Based on the equation shown in chapter 2.6.3 the transient behaviour and steady state concentration are
calculated by some of the metabolites that have not been measured, see appendix E. The transient
behaviour of the normalized cofactor ratio are presented in Figure 3.2. In Table 3.13 the steady state
concentrations are listed.
AcCoA/HCoA
NADH/NAD
6.00
3.00
4.00
2.00
2.00
1.00
-
-
succCoA/HCoA
4.00
3.00
2.00
-100
0
100
200
300
400
500
-100
1.00
-
0
100
[sec]
NADPH/NADP
2.50
2.00
1.50
1.00
0.50
-100
200
300
400
500
-100
100
200
[sec]
300
400
500
-100
100
200
[sec]
THF/MYTHF
THF/METHF
1.50
1.50
1.00
1.00
0.50
0.50
-
0
0
[sec]
300
400
500
300
400
500
0
100
200
300
400
[sec]
500
-100
0
100
200
[sec]
Figure 3.2: Normalized transient behaviour of cofactor ratios
50
Table 3.13: Steady state concentrations
metabolite
mmol/gDw
metabolite
mmol/gDw
O2:cyt
0.627 AcCoA:cyt
CO2:cyt
0.672 HCoA:cyt
GAP:cyt
0.069 succCoA:cyt
3PG:cyt
0.546 FAD:cyt
1.508
NADH:cyt
0.387 FADH2:cyt
0.792
NAD:cyt
1.913 NADPH:cyt
0.726
Xylu5P:cyt
0.150 NADP:cyt
1.574
Rib5P:cyt
0.096 THF:cyt
9.138E-05
Ribu5P:cyt
0.080 METHF:cyt
2.681E-03
E4P:cyt
0.013 MYTHF:cyt
2.30
sed7P:cyt
0.439 ExPept:cyt
170
OAA:cyt
0.001 psacch:cyt
306
citr:cyt
2.178 PAA:cyt
7.81
iCitr:cyt
0.090 penG:cyt
3.23
Ratios
-
0.00457
2.288
0.00729
-
FADH2/FAD
0.526 NADH/NAD
0.202
AcCoA/HCoA
0.002 succCoA/HCoA
0.003
NADPH/NADP
0.461 THF/MYTHF
METHF/MYTHF
0.00004
0.0012
Note that even though the results are given in μmol/gDW, the calculations have been performed on the
basis of mol/l to avoid dimension problems with respect the equilibrium constants.
3.2.4 Checking PEQ assumptions
Now that all necessary metabolites are know, it is checked whether the mass action ratios of those
reactions in Table 2.1 are close equilibrium. In other words, the calculated profiles of NADH/NAD,
AcCoA/HCoA etc, are used to calculate the mass action ratio to check whether these are consistent with
other reactions that may be candidates for PEQ. The average mass action ratio during the time frame of
the pulse experiment, written as Kav, is calculated as product/substrate as defined in Table 2.1 and
presented in Table 3.14 and compared with the equilibrium constants from literature, Klit. Note that the
51
calculated mass action ratios are based on the average concentration of a metabolite of the whole cell and
may be therefore different from the value reported in literature. The fourth column of this table the ratio
between the standard deviation and the average mass action ratio is given, which if the percentage is high,
indicates a high variance in the calculated mass action ratio.
The calculated mass action ratio of aspartate transaminase (r11.16) is incomparable to the value found in
literature, probably caused by the fact that the calculated OAA is too high. OAA has been calculated based
on malate dehydrogenase reaction (r4.11), which is part of the C4 equilibrium pool. Also the calculated
mass action rate of glutamate-ammonia ligase (r11.2) is significantly different from the value reported in
literature which might be caused by the fact that the used NH4/Pi ratio is wrong. The intracellular
concentration of Pi is assumed to be 10 mM (Teusink et al. 2000) and the NH4 concentration 0.1 mM.
These concentrations were chosen in order to get reasonable ratios of NADH/NAD and NADPH/NADP.
Increasing the NH4 extent by a factor 10 will lead that the calculated mass action ratio of r11.2 comes
closer to the reported value of 275, but it also lead to the result that almost no NADP is available in the
cell which is physiological unlikely.
Table 3.14: Average mass action ratios based on data
Reactions
r1.2
l1.3
r4.10
r5.1
r8.1
r11.2
r11.16
r11.20
r11.22
l17.3
r13.6
Kav
0.19
0.318
4.856
3.56E-03
8.04E-04
11.4
1132
4.30E-03
0.924
0.161
2.38E-03
stdev
0.02
0.066
0.122
8.41E-04
4.51E-04
2.456
410
2.55E-03
0.179
0.019
5.21E-04
stdev/Kav
9.8%
20.9%
2.5%
23.6%
56.1%
21.5%
36.2%
59.3%
19.4%
12.0%
21.9%
Klit
0.33 (Staples, J.F. et al., 1997)
0.42 (Lowry, O.H. et al., 1964) &
4.60 (Wold, F et al, 1957)
N/A
N/A
275
6.76 (Krebs et al, 1953)
N/A
1.28 (Tewari et al, 1998)
0.06 (Guynn, R.W. et al 1974)
N/A
Despite having problems finding reasonable values for concentrations of necessary but unmeasured
metabolites like NH4 and Pi, the normalized profiles during the pulse remain unchanged since it is
assumed that the concentrations of phosphate and ammonia are constant. Hence the calculated mass action
ratios shown in may be a bit off target with respect to its absolute value, but the normailized transient
behaviour will be independent by the chosen concentrations of NH4 and Pi. From Figure 3.3 a clear
dynamic response can be recognized for pyruvate carboxylase (r5.1), glycine cleavage system (r8.1),
aspartate transaminase (r11.16), alanine transaminase (r11.22), ATP hydrolysis (r9.1) and CTP synthase
(r13.6). The remaining calculated mass action ratios seem to be reasonable constant.
52
l1.3
r1.2
0.200
5.000
0.100
0.200
0.000
4.700
4.600
0.000
0
100
200
300
400
500
-100
4.500
0
100
Time (sec)
500
-100
0.000
16.000
0.001
12.000
200
300
400
500
-100
200
300
400
-100
500
200
r11.22
300
400
500
-100
200
300
400
-100
500
0
100
0.2
0.0030
0.16
0.0025
400
500
0.0020
K (-)
K (-)
300
r9.1
0.08
0.0015
0.0010
0.0005
0.0000
0
Time (sec)
200
Time (sec)
0.12
-100
500
0.000
100
0.04
500
400
0.600
l17.3
400
300
0.800
Time (sec)
300
500
0.200
0
r13.6
200
400
0.400
Time (sec)
0.0035
0.003
0.0025
0.002
0.0015
0.001
0.0005
0
300
1.000
K (-)
K (-)
200
500
1.200
0.007
0.006
0.005
0.004
0.003
0.002
0.001
0.000
0
100
100
r11.20
400
0
0
r11.16
800
100
100
Time (sec)
1200
400
0.000
0
Time (sec)
1600
300
4.000
Time (sec)
2000
0
200
8.000
0.000
100
100
r11.2
0.002
0.000
0
0
Time (sec)
0.001
0.002
K (-)
400
K (-)
K (-)
K (-)
0.004
K (-)
300
r8.1
r5.1
-100
200
Time (sec)
0.006
-100
4.800
0.100
0.050
-100
4.900
K (-)
K (-)
K (-)
5.100
0.300
0.150
-100
r4.10
0.400
0.250
0
100
200
300
400
500
-100
0
Time (sec)
100
200
Time (sec)
Figure 3.3: Calculated mass actions reactions of candidates of PEQ listed in Table 2.1.
Based on Figure 3.3 the following rates can be assumed to close to equilibrium:
Table 3.15: Reactions assumed to be close to equilibrium
Glycolysis
1
r1.2
g6P = f6P
2
r1.4
f16P = 2 GAP
3
l1.2
GAP+NAD+Pi+ADP = 3PG + ATP + H + NADH
4
l1.3
3PG = H2O + PEP
Pentose phosphate pathway
5
r2.3
Ribu5P = Rib5P
6
r2.4
Ribu5P = Xylu5P
53
7
r2.5
Rib5P + Xylu5P = GAP + sed7P
8
r2.6
GAP + sed7P = E4P + f6P
9
r2.7
E4P + Xylu5P = f6P + GAP
TCA cycle
10
r4.2
AcCoA + H2O + OAA = citr + H + HCoA
11
r4.3
citr = iCitr
12
r4.8
ADP + Pi + succCoA = ATP + HCoA + succ
13
r4.9
FAD + succ = FADH2 + fum
14
r4.10
fum + H2O = mal
15
r4.11
mal + NAD = H + NADH + OAA
Transport of 1-C Compounds
16
r8.2
H + METHF + NADH = MYTHF + NAD
Transport across plasma membrane
17
r9.6
O2:ext = O2:cyt
18
r9.7
CO2:cyt = CO2:ext
19
r9.15
H2O:cyt = H2O:ext
Amino Acid synthase
20
r11.1
aKG + H + NADPH + NH4 = glu + H2O + NADP
21
r11.10
ser + THF = gly + H2O + METHF
22
r11.20
homcys + MYTHF => met + THF
Synthesis of glycogen and polysaccharides
23
l17.3
g6P = g1P
3.2.5 Elimination of PEQ rates and new defined equilibrium pools
Now that the pseudo equilibrium reactions have been identified the stoichiometric model can be reduced
by the metholody discussed in chapter 2.6.5.
The following equilibrium pools can be derived from the pool composition matrix, which is shown in
appendix D.
p1 = [ aKG ] − [ NH 4 ]
(4.49)
p2 = [ glu ] + [ NH 4 ]
(4.50)
p3 = [ ATP ] + [ Pi ]
(4.51)
p4 = [ ser ] + [ METHF ] + [ MYTHF ] − [ homcys ]
(4.52)
54
p5 = [ AcCoA] +
1
1
[ H ] + [ Pi ]) − ([ ser ] + [ METHF ] + [ succCoA] + [OAA] + [ NH 4 ])
(
2
2
(4.53)
p6 = [ NADH ] +
1
1
[ NH 4 ] + [ Pi ]) − ([ ser ] + [ METHF ] + [ succCoA] + [OAA] + [ H ])
(
2
2
(4.54)
p7 = [ succCoA] + [ succ ] + [ FADH 2 ]
(4.55)
p8 = [ NADPH ] − [ NH 4 ]
(4.56)
p9 = [ ser ] + [ gly ]
(4.57)
p10 = [3PG ] + [ PEP ] + [ Pi ] − [ succCoA]
(4.58)
p11 =
1
1
1
1
1
[ Rib5P] +[ Ribu5P] +[ Xylu5P]) + [ E4P] + ([GAP] +[ succCoA]) +[ f 16P] − [ sed7P] − [ Pi]
(
6
3
2
6
2
p12 = [ homcys ] + [ met ]
(4.59)
(4.60)
p13 =
1
1
[ H] + [ Pi] + [OAA] + [ succCoA]) − ([ NH4 ] + [ METHF] + [ ser]) + [ succ] + [mal] + [ fum]
(
2
2
(4.61)
p14 =
1
1
[ NH4 ] + 0.5[OAA] + 0.5[ succCoA] + 0.5[ METHF] + 0.5[ ser]) − ([ H] +[ Pi]) +[iCitr] +[citr]
(
2
2
(4.62)
1
2
4
[ E 4P] + ([ Rib5P] + [ Ribu5P] + [ Xylu5P]) + [ sed 7P] + [ f 6P] + [ g1P] + [ g6P]
3
3
3
(4.63)
p15 =
p16 = [ H ] + [ Pi ] + [ H 2O ] + [ mal ] + [ H 2O : ext ] − [ PEP ] − [ succCoA] − [ METHF ]
(4.64)
p17 = [O2 ] + [O2 : ext ]
(4.65)
p18 = [CO2 ] + [CO2 : ext ]
(4.66)
As can be seen, the pool composition matrix contains several negative elements, which can be removed by
lumping several of the above mentioned equilibrium pools into a new pool. The following equilibrium
pools are lumped:
plump1 = p1 + p2
(4.67)
plump 2 = p4 + p5 + p6 + p12 + p13 + p14
(4.68)
plump 3 = p2 + p8
(4.69)
plump 4 = p7 + p10 + p11 + p15
(4.70)
plump 5 = p4 + 2 ⋅ p7 + p10 + p11 + p12 + p16
(4.71)
This results in the following lumped equilibrium pools
plump1 = [ aKG ] + [ glu ]
(4.72)
55
plump2 = [ Pi ] + [ citr] + [iCitr] + [ succ] + [ mal ] + [ fum] + [ met ] + [ MYTHF ] + [ AcCoA] + [ NADH ] (4.73)
plump 3 = [ glu ] + [ NADPH ]
plump4 = [ f 6P] + [ g1P] + [GAP] +
(4.74)
1
5
[ Pi] + [ succCoA]) + ([ Rib5P] + [ Ribu5P] + [ Xylu5P]) ...
(
2
6
7
2
+ [ sed 7P] + [ E4P] + [ PEP] + [ succ] + [ FADH2 ] + [ 3PG] + [ f 16P] + [ g6P]
6
3
(4.75)
plump5 = [ H] + 2[ Pi] +[ H2O] + 2[ succ] +[ mal] +[ ser] +[ MYTHF] +[ FADH2 ] +[3PG] +[ met] +[ H2O: ext] (4.76)
The new pool composition matrix does now include these lumped equilibrium pools equations together
with the unused equilibrium pools p3, p9, p17, p18 and is subsequently multiplied by the submatrix Sv_peq
which results in the submatrix Sp, see equation (2.83). The pool composition matrix has a rank of 9 and is
therefore not rank deficient in the rows. In other words, the above mentioned pool equations are
independent. In the independent stoichiometric matrix S, the subsystem Sv_peq is replaced by Sp. The
columns representing the PEQ rates, which have been presented in Table 3.15, contain zero entries and
can subsequently be omitted from the stoichiometric matrix.
The resulting reduced stoichiometric matrix, written as Sred, has been reduced to a 51 x 52 matrix which is
the result of eliminating 23 reactions and lumping 41 metabolites that are involved in PEQ reactions into 9
equilibrium pools.
3.2.6 Turnover times
In this chapter the turnover times are presented, which has been calculated with equation (2.69) on the
basis of the metabolic fluxes of the penGv4 model.
In Table 3.16 the calculated turnover times are presented in ascending order with respect to the turnover
time. In the last column the turnover times presented in (Nasution 2007) are shown and it can be seen that
despite the similar experimental conditions, many dissimilarities exists. Especially for t6P, aAd and ala
large differences exist. This difference must be caused by the calculated of the net influx, due to the fact
that the available datasets are comparable, see appendix A.
The dissimilarity in τ with respect to aAd is caused by the fact that the whole penG biosynthesis has been
lumped into one reaction. Since aAd is consumed but also produced in this synthesis, there is no net flux
for aAd. Based on the original stoichiometric model (van Gulik et al. 2000), a turnover rate for aAd of 299
sec is calculated which resembles the value from Nasution.
56
Table 3.16: Turnover times measured metabolites
X
metabolite
Jin
[μmol/gDW.s] [μmol/gDW]
τ
τNasution
[s]
[s]
ADP:cyt
2.27
1.061
0.47
PEP:cyt
0.240
0.191
0.80
0.9
pyr:cyt
0.232
0.471
2.03
0.9
6Pgluct:cyt
0.094
0.278
2.94
3.7
ATP:cyt
2.27
7.345
3.24
succ:cyt
0.117
0.481
4.11
3.3
f16P:cyt
0.114
0.675
5.93
7.2
aKG:cyt
0.196
1.188
6.05
22.1
fum:cyt
0.123
0.767
6.25
13
f6P:cyt
0.119
0.814
6.83
5.7
g1P:cyt
0.0347
0.728
21.00
g6P:cyt
0.191
4.546
23.79
23.3
cys:cyt
0.00364
0.097
26.50
18
mal:cyt
0.123
3.748
30.52
19
GMP:cyt
0.00069
0.022
32.00
met:cyt
0.00219
0.137
62.66
58.8
phe:cyt
0.00235
0.191
81.19
61.2
ile:cyt
0.00245
0.370
150.72
111.3
homcys:cyt
0.00219
0.370
169.16
375.6
leu:cyt
0.00400
0.732
183.12
131
trp:cyt
0.000533
0.108
203.19
130
tyr:cyt
0.00127
0.264
208.42
144.4
val:cyt
0.00784
2.076
264.67
243
gly:cyt
0.00741
2.082
280.98
243.6
pro:cyt
0.00313
0.949
303.15
205.5
0.000920
0.282
306.54
ser:cyt
0.0142
5.695
402.01
453.2
lys:cyt
0.00252
1.243
492.77
355.8
his:cyt
0.00131
0.716
547.01
432
glu:cyt
0.0893
52.959
592.86
657.5
AMP:cyt
57
asn:cyt
0.00215
1.477
687.33
458.7
aAd:cyt
0.00252
1.811
717.69
292.8
asp:cyt
0.0175
16.273
927.31
717
orn
0.00325
3.056
940.78
t6P:cyt
0.00053
0.542
1026.81
47.8
thr:cyt
0.00566
5.933
1048.29
757.5
gln:cyt
0.0179
28.700
1606.63
1243
CTP:cyt
0.000443
0.956
2159.66
UTP:cyt
0.000443
1.361
3074.95
0.00669
21.676
3242.23
ala:cyt
269.1
This illustrates that lumping reactions may cause a severe overestimation of the turnover times if the
metabolic flux is high. Even in the original metabolic model there are several lumped reactions that have
no net flux for some metabolites, e.g. aKG, despite the fact that these are involved the biosynthesis. Hence
the actually net flux in might be higher which results that the calculated turnover time based on a lumped
metabolic network might be higher than that of an unlumped metabolic network. One may therefore
expect that the high turnover time of ala and t6P can be contributed to lumping but calculation based on
the unlumped network shows that this is not the case.
The time scale of the pulse response experiment performed by Uly Nasution is 420 sec and her results
showed that most metabolites reached a pseudo steady state level after 180 second. Therefore one would
expect that metabolites with are turnover time significantly higher than that, lets say arbitary 2 times the
last made measurement, i.e >820 sec, can be considered to be frozen. The metabolites asp, orn, t6P, thr,
gln, CTP, UTP, ala can be considered as frozen pools based on the results of Table 3.16. However the
normalized concentration profiles that are shown in appendix A, only suggest that asp, orn and UTP are
frozen. It also shows that t6P is clearly very dynamic and illustrates that identification of frozen pools on
the basis of the turnover time should be used with caution. Although turnover times may give an
indication, a transient behaviour should be checked in order to determine whether a metabolite is frozen,
dynamic or at pss.
Metabolites that can be considered to be at pseudo steady state are discussed in the next chapter.
58
3.2.7 PSS metabolites
The pseudo steady state assumption for a particular metabolite allows the removal of its differential
equation. A pseudo steady state metabolite has a very short transient behaviour are which it reaches the
pseudo steady state.
The pseudo steady state assumption should not be applied for allosteric effectors (Visser et al. 2000), such
as ADP and ATP on phosphofructokinase and pyr on pyruvate decarboxylase.
Many metabolites that, based on their turnover time, can be considered to be a pss metabolite, e.g. succ
and fum, are part of the equilibrium pools have discussed in chapter 3.2.5. However 6Pgluct has not and is
therefore target for further reduction. Recall equation (2.88), which gives the normalized concentration in
a logarithmic space of 6Pgluct as a linear relation of the kinetic parameters a1, a2 and a3, as defined in
equation (2.89) can be estimated by linear regression, which has been shown in Table 3.17. The estimation
results seems to be fitted nicely at first glance based on a reasonable R2 of 0.97 and a error variance of
4.3*10-3. However a closer look reveals that the standard error, written as SE, is relatively high compared
the estimated values for ATP and also NADPH. The shown t-value is equal to:
t = α j / SE (α j )
(4.77)
and has a Student t-distribution with n-1 degrees of freedom. With the t-value the hypothesis can be tested
whether bj is zero and should be omitted. As a rule of thumb, if |t|>2, xj might be considered as a candidate
to omit from the model. The VIF is the variance inflation factors (VIFs) and as a rule of thumb, if VIF>10,
xj might be a candidate for removal (Lindfeld and Penny 1999).
Table 3.17: Estimated pss parameters, R2=0.97
X
SE
t-value VIF
α
g6P
0.77
0.16
3.94
4.16
ATP
-0.19
0.31
-0.62
2.63
NADPH
-0.42
0.16
-2.70
1.21
Hence ATP is a candidate to omit from the estimation process and the the result is shown in Table 3.18.
The R2 has not changed but the error variance has decreased a bit to 3.6*10-3. However the standard error
has been decreased significantly suggesting a better fit has been obtain than in Table 3.17.
59
Table 3.18: Estimated pss parameters, R2=0.97
X
SE
t-value VIF
α
g6P
NADPH
0.88
0.07
12.84
0.61
-0.35
0.09
-3.68
0.53
For practical reasons often unmeasured metabolites are treated as pss metabolites, which is in fact already
largely been done by the data-driven reduction of the metabolic network. Metabolites that are still left in
the stoichiometric matrix but have not been measured are ExPept, arg, SO4, penG, PAA, psacch and tre,
and their response is unknown. The responses of penG, PAA or any intermediate in the penicillin
biosynthesis such as iPN are unknown and any assumption regarding their behaviour would be purely
speculative. Nasution also revealed that the dynamics of non-penicillin related amino acids is relatively
small and hence ExPept, arg and the related SO4, substrate for homcys and cys, will be treated as pss
metabolites.
However the perturbation experiments of Nasution (Nasution et al. 2006) revealed that due to the limited
increase of oxygen uptake during the glucose pulse, a significant part of the abundant glucose is converted
into storage material. And as consequence assuming that storage materials such as tre and psacch are at
pseudo steady state may not hold. The dynamics of these storage materials can not be simulated because
the kinectic parameters are unknown.
3.2.7.1 Removal of pss metabolites
The pss metabolites and the corresponding metabolites have been ordered to top left bottom of the
stoichiometric matrix and form the submatrix Spss. Subsequently the independent reactions have to chosen,
which has been done in such a way that the lower part of the null space of Spps form an identity makes. In
other words the independent reaction rates are organized at the right of Spps.
There are 6 pss metabolites which are involved in 12 reactions. The choice for independent reactions is the
penicillin biosynthesis (l19.1), biomass formation reaction (l18.2), sulfate uptake (r9.4), cys biosynthesis
(l11.7), Ribu5P biosynthesis (r2.1) and the excretion of peptides (r9.13).
After calculation of Slump and subsequently replacing the subsmatrix Sv_pss, the pss metabolites only
contains zero entries and hence can be omitted. The stoichiometric matrix is reduced by 6 metabolites and
6 rates and has now the dimension of 45x46.
60
3.2.8 Frozen Metabolites
Metabolites that have been assumed to be constant are NH4:ext, SO4:ext, H:ext, Pi:ext, which reduces the
number of differential equations with 4. In Table 3.19 a summary is given from the results of the timescale analysis. This has allowed the system to be reduced half its size to a 41x46.
Table 3.19: Results of the Time-Scale Analysis
PEQ
f6P:cyt , g1P:cyt, GAP:cyt, H:cyt, CO2:cyt, Pi:cyt, H2O:cyt, Rib5P:cyt, Ribu5P:cyt,
Xylu5P:cyt, sed7P:cyt, E4P:cyt, PEP:cyt, NH4:cyt, OAA:cyt, iCitr:cyt, succCoA:cyt,
succ:cyt, homcys:cyt, mal:cyt, METHF:cyt, O2:cyt, ser:cyt, aKG:cyt, glu:cyt,
ATP:cyt, MYTHF:cyt, AcCoA:cyt, NADH:cyt, FADH2:cyt, NADPH:cyt, gly:cyt,
3PG:cyt, f16P:cyt, met:cyt, fum:cyt, citr:cyt, g6P:cyt, H2O:fer, O2:fer, CO2:fer
PSS
6Pgluct:cyt, ExPept:cyt, SO4:cyt, arg:cyt, penG:cyt, PAA:cyt
Frozen
NH4:ext, SO4:ext, H:ext, Pi:ext,
Dynamic
plump1, plump2, plump3, plump4, p3, p9, pyr:cyt, aAd:cyt, lys:cyt, ile:cyt, thr:cyt, ala:cyt,
asn:cyt, asp:cyt, cys:cyt, gln:cyt, his:cyt, leu:cyt, phe:cyt, pro:cyt, trp:cyt, tyr:cyt,
val:cyt, CTP:cyt, UTP:cyt, t6P:cyt, orn:cyt, GMP:cyt, AMP:cyt, psacch:cyt, tre:cyt,
plump5, p17, p18, glc:ext, psacch:ext, penG:ext, PAA:ext, tre:ext, biomass:ext,
ExPept:ext
3.3 Postulation of Reaction Kinetics
In this chapter the chosen regulators of the irreversible reactions, which are summarized in Table 3.20, are
discussed.
3.3.1 Glycolysis
l1.1: ATP + H:ext + glu:ext => ADP + 2H + g6P
Since the intracellular glucose concentration in the cytosol is assumed to be very low, the glucose
transport across the plasma membrane and the hexokinase catalyzed reaction (r1.1) are lumped.
Hexokinase is activated by glucose and inhibited by its product g6P. Citrate acts as a noncompetitive
inhibitor with respect to glc and is further regulated by the ATP/ADP rate and the feedback inhibitor t6P
(Brenda 2006).
61
r1.3: ATP + f6P => ADP + f16P + H
6-Phosphofructosynthase is activated by f6P and inhibited by its product f16P and citr (Brenda 2006).
AMP activates at low f6P concentrations but starts to inhibit phosphofructosynthase at high
concentrations. Furthermore the activity is regulated by the ATP/ADP ratio. Other found effectors are the
activators g6P, NH4, Pi and the inhibitor PEP but these will be neglected as these don’t appear to have a
significant regulating role (Visser et al. 2004b).
r1.9: ADP + PEP + H => ATP + pyr
Pyruvate kinase is activated by PEP and f16P and is moreover regulated by the ADP/ATP ratio and by
feedback inhibition of citr (Brenda 2006).
3.3.2 Pentose phosphate pathway
r2.1: g6P + H2O + NADP => 6Pgluct + 2 H + NADPH
This reaction is catalyzed by three enzymes. Initially glucose-6-phosphateisomerase isomerizes α-glc into
β-glc after which glucose-6-phosphate-1-dehydrogenase catalyses the irreversible conversion of β-glc into
D-glucose-1,5-lactone-6-phosphate. This enzyme is activated by glc (Brenda 2006) and inhibited by
NADPH and ATP (Vaseghi et al. 2001). The final conversion is catalyzed by 6-phosphogluconolactonase
in which D-glucose-1,5-lactone-6-phosphate reacts with water to synthesize 6Pgluct.
r2.2: 6Pgluct + NADP => CO2 + NADPH + Ribu5P
Phosphogluconatedehydrogenase is activated by its substrate 6Pgluct and inhibited by NADPH (Brenda
2006) and ATP (Vaseghi et al. 2001). Furthermore the enzyme is inhibited by f16P, Pi, Ribu5P, SO4,
UTP and activated by f6P although none of these metabolites incorporated in Vaseghi’s model of the PPP.
Hence their regulatory effect will be neglected for now.
62
3.3.3 TCA cycle
r4.1: HCoA + NAD + pyr => AcCoA + CO2 + NADH
The oxidative decarboxylation to synthesize AcCoA from pyr is catalyzed by the pyruvate dehydrogenase
complex (PDC), which consists of three enzymes denoted as E1 (pyruvate dehydrogenase), E2
(dihydrolipoamide S-acetyltransferase) and E3 (dihydrolipoamidedehydrogenase), see Figure 3.4. The rate
limited step within this complex is the reaction catalyzed by pyruvate dehydrogenase in which pyr
decarboxylates due to the reaction with the involved cofactor TPP (thiamine pyrophosphate). The
removed acetyl-group attaches to the cofactor TPP through a thioester bond. Subsequently pyruvate
dehydrogenase transfers the acetyl group and two electrons to an oxidized lipoic acid to which it becomes
esterified. Dihydrolipoamide S-acetyltransferare catalyzed the trans-esterification from the sulfide of the
lipoyl to HCoA after which AcCoA is produced. The reduced lipoic acid is re-oxidized by concomitant
reduction of FAD that is present in E3. The formed FADH2 subsequently reduces NAD which results that
PDC returns to its original redox state.
Figure 3.4: Mechanism of pyruvate dehydrogenase complex (PDC)
63
According to the Brenda database (Brenda 2006) E1 is activated by pyr and E3 is activated by NAD.
Furthermore it is assumed that E1 is subject of (end) product feedback inhibition of CO2 and AcCoA. Due
to the near equilibrium intermediate reactions, the corresponding absolute values of the elasticities are
identical. In addition citr is inhibiting E1 (Brenda 2006)
In addition the PDC is inhibited if the ratios ATP/ADP, NADH/NAD and AcCoA/HCoA are increased
(Voet and Voet 2004).
r4.4: iCitr + NAD => aKG + CO2 + NADH
Isocitrate dehydrogenase (NAD+) is activated by its substrate by iCitr and further regulated by the
NAD/NADH ratio. OAA and citr slightly inhibiting isocitrate dehydrogenase and will therefore be
neglected.
r4.7: aKG + HCoA + NAD => CO2 + NADH + succCoA
This reaction is catalyzed by the oxoglutarate dehydrogenase complex (ODC) which works much like the
pyruvate dehydrogenase complex. It is composed of the enzymes, oxoglutarate dehydrogenase,
dihydrolipolipoamide S-succinyltransferase and dihydrolipoamide dehydrogenase that employ coenzymes
TPP, lipoic acids and FAD respectively.
ODC is inhibited by its products succCoA, CO2 and NADH (Voet and Voet 2004) and activated by aKG
(Brenda 2006). Furthermore it is subject to feedback inhibition of OAA.
3.3.4 Anaplerotic pathways
r5.1: ATP + CO2 + H2O + pyr => ADP + Pi + OAA
This reaction is catalyzed by pyruvate carboxylase which is activated by CO2 and pyr. In the presence of
asp and aKG it is activated by AcCoA. Furthermore asp is an allosteric inhibitor (Brenda 2006).
3.3.5 Oxidative phosphorylation
The oxidative phosphorylation has been dealt in chapter 3.2.2. Reaction l6.7 is activated by FADH2 and
O2 and inhibited by ATP. The elasticities are linked with the stoichiometric coefficients, i.e. 1.104
64
εl6.1,FADH2 = εl6.1,ATP. Reaction l6.8 is analogously regulated as l6.7, although in this case NADH is the
activator instead of FADH2. Again the corresponding elasticities are related by their stoichiometric
coefficients.
3.3.6 Transfer of 1-C compounds
r8.1: gly + NAD + THF => CO2 + METHF + NADH + NH4
No effectors have been identified for this reaction. It will be assumed that this reaction is activated by gly
and NAD and inhibited by METHF.
3.3.7 Transport across plasma membrane
In this chapter the transport across the plasma membrane are discussed. These reactions can not be
considered as close to equilibrium since they are either driven by active transport or consist of the
transport of large molecules such as ExtPept, psacch and penG.
Due to the fact that the concentration ratio across the plasma membrane is unknown and that some
metabolites are not measured, the corresponding elasticities can not be determined. As a consequence the
elasticities will be set to zero which implies that the transport rate will remain constant. The transport
steps are:
r9.2: 2 H:ext + Pi:ext => 2 H:cyt + Pi:cyt
r9.4: 2 H:ext + SO4:ext => 2 H:cyt + SO4:ext
r9.8: penG:cyt=> penG:ext
r9.11: PAA:ext => PAA:cyt
r9.13: ExPept:cyt => ExPept:ext
r9.14: psacch:cyt => psacch:ext
r9.20: H:ext + NH4:ext => H:cyt + NH4
3.3.8 Amino acid synthesis
Frequently the first reaction in the biosynthesis of an amino acid is irreversible which is catalyzed by an
enzyme that is positively activated by its substrate and is subject of (end) product feedback inhibition.
This is called the committed step.
65
r11.2: ATP + glu + NH4 => ADP + Pi + gln + H
This reaction is catalyzed by glutamate-ammonia ligase for which no effectors have been found. It will be
assumed that this reaction is activated by glu and inhibited by gln.
r11.3: ATP + glu + 2 H + 2 NADPH => ADP + H2O + 2 NADP + Pi + pro
The proline biosynthesis consists of 3 intermediate conversions which, in chronological order, are
catalyzed by glutamate 5-kinase, glutamate-5-semialdehyde dehydrogenase and pyrroline 5-carboxylate
reductase. Only the first reaction catalyzed by glutamate 5-kinase is irreversible. Glutamate 5-kinase is
positively activated by glu and ATP but is negatively regulated by feedback inhibition of pro.
r11.7: AcCoA + glu + H2O + NAD => aAd + CO2 + HCoA + NADH
The biosynthesis of aAd is catalyzed, in chronological order, by homocitrate synthase, homoaconitate
hydratase, homoisocitrate dehydrogenase and 2-aminoadipate transaminase. Only the first reaction
catalyzed by homocitrate synthase is irreversible. Homocitrate synthase is activated by AcCoA, aKG and
inhibited by the product aAd and end product lys. Other found regulators such as the activator ATP and
the inhibitors AMP and HCoA are assumed to have a neglectable influence.
r11.8: aAd + 2 ATP + glu + H2O + NAD + 2 NADPH => 2 ADP + aKG + H + lys + NADH + 2
NADP + 2 Pi
The biosynthesis of lys is catalyzed, in chronological order, by L-aminoadipate-semialdehyde
dehydrogenase, saccharopine dehydrogenase and saccharopine dehydrogenase. Only the first intermediate
reaction is irreversible. Aminoadipate-semialdehyde is positively activated by aAd and is subject of
product inhibition of lys. Other regulators found for aminoadipate-semialdehyde are the activator ATP and
the competitive inhibitors asp and glu. Although glu is consumed it is being converted into aKG which in
equilibrium with glu. As a consequence it will be assumed glu has a neglectable regulatory role. The
function of asp on the lys biosynthesis is unclear since both synthesis don’t seem to be related and there its
influence on the activity will be assumed to be neglectable. The amount of ATP consumed is relatively
low compared to the homcys, cys or arg biosynthesis and to keep the amount of non-zero elasticities to a
minimum it will be assumed the elasticity of ATP for this reaction can be set to zero.
66
r11.9: 3PG + glu + H2O + NAD => aKG + H + NADH + Pi + ser
This reaction consists of three intermediate conversions that are catalyzed, in chronolotical order, by
phosphoglycerate dehydrogenase, phosphoserine transaminase and phosphoserine phosphatase. Only the
final intermediate conversion is irreversible and the involved enzyme phosphoserine phosphatase is
activated by PHser and is subject of feedback inhibition by ser. Although PHser is not included in the
stoichiometric model its regulatory role can be expressed by 3PG, glu, aKG and the NAD/NADH ratio
due to the fact that the first two conversions are near equilibrium. As a consequence all resulting non-zero
elasticties for this reaction have identical values. No effectors have been found for phosphoserine
phosphatase.
r11.16: glu + OAA => aKG + asp
No effectors have been identified for aspartate transaminase. It will be assumed this reaction is activated
by OAA and inhibited by asp.
r11.17: asp + 2 ATP + H2O + NH4 => 2 ADP + asn + 2 H + 2 Pi
The biosynthesis of asn consists of one irreversible reaction and is catalyzed by aspartate-ammonia ligase,
which is positively activated by asp and is subject of feedback inhibition by asn. The regulatory role of the
ATP/ADP ratio will be assumed to be neglected.
r11.21: glu + 2 H + NADPH + pyr + thr => aKG + CO2 + H2O + ile + NADP + NH4
The biosynthesis of ile contains 5 intermediate reaction step. These are catalyzed, in chronological order,
threonine amino-lyase, acetolactate synthase, ketol-acid reductoisomerase, dihydroxy-acid dehydratase
and branches-chain-amino-acid trasnaminase. Only the second intermediate reaction catalyzed by
acetolactate synthase is irreversible. The enzyme is positively activated by pyr and subject to end product
feedback inhibition by val and ile.
r11.22: glu + pyr => aKG + ala
Alanine transaminase is activated by pyr and inhibited by ala.
67
r11.34: 0.134 ala + 0.07 arg + 0.0386 asn + 0.0386 asp + 0.02 cys + 0.0509 gln + 0.0509 glu + 0.132 gly
+ 0.0119 his + 0.0307 ile + 0.0559 leu + 0.0348 lys + 0.0103 met + 0.0266 phe + 0.0537 pro + 0.044 ser
+ 0.0467 thr + 0.01 trp + 0.0203 tyr + 0.12 val => 4.53 ExPept + H2O
It will be assumed that all elasticities are zero which results that the reaction remains at steady state.
l11.1: 2 H + NADPH + glu + 2 pyr => CO2 + H2O + NADP + aKG + val
This reaction is the result of the lumping the aKI biosynthesis (r11.23) with the valine biosynthesis
(r11.24). The aKI biosynthesis consists of the catalysis of acetolactate synthase, ketol-acid
reductoisomerase and dihydroxy-acid dehydratase. The val biosynthesis is catalyzed by branched-chainamino-acid-transaminase. These enzymes also involved in the biosynthesis of leu (r11.21) and as a
consequence also the valine biosynthesis is activated by pyr and inhibited by ile and its product val.
l11.2: AcCoA + H + NADPH + NAD + glu + 2 pyr => 2 CO2 + HCoA + NADH + NADP + aKG + leu
This reaction is the result of lumping the aKI biosynthesis (r11.23), bIM biosynthesis (r11.25) and leu
biosynthesis (r11.26). The first synthesis has already been discussed in reaction l11.1 in which pyruvate is
an activator and val and ile are product feedback inhibitors.
The bIM biosynthesis consists of two intermediate reactions that are, in chronological order, catalyzed by
2-isopropylmalate synthase and 3-isopropylmalate dehydratase. Only the first reaction is irreversible and
the involved enzyme is subject to feedback inhibition by leu. Furthermore it is activated by AcCoA. The
metabolite phe is also found to inhibit 2-isopropylmalate synthase at a concentration of 10 mM. Since the
physiological concentration in the cell of phe is much lower, it will be assumed that its regulatory
influence is neglected.
The biosynthesis of leu from bIM is catalyzed by 3-isopropylmalate dehydrogenase and is reversible.
l11.3: 3 ATP+E4P+NADPH+2 PEP+Rib5P+gln+ser=>3 ADP + CO2 + GAP + H2O + 2 H + NADP +
6 Pi + glu + pyr +trp
l11.4: ATP+E4P+NADPH+NAD+2 PEP+glu=>ADP+CO2+NADH+NADP+4 Pi+aKG+tyr
l11.5: ATP+E4P+H+NADPH+2 PEP+glu=>ADP +CO2 + H2O + NADP + 4 Pi+aKG+phe
68
The biosynthesis of trp, tyr and phe are very similar as they all utilize the chor biosynthesis (r11.27).
However since chor has not been measured it has been eliminated from the stoichiometric model by
lumping r11.27 with r11.30 & r11.31, r11.29 and r11.28 which results in the above listed reactions for trp
synthesis (l11.3), tyr (l11.4) and phe (l11.5).
The chor biosynthesis is, in chronological order, catalyzed by 3-deoxy-7-phosphoheptulonate synthase, 3deoxy-D-arabino-heptulosanate-7-phosphate, 3-dehydroquinate dehydratase, shikimate dehydrogenase,
shikimate kinase, 3-phosphoshikimate 1-carboxyvinyltransferase and finally by chorismate synthase. Only
the reaction catalyzed by 3-deoxy-7-phosphoheptulonate synthase is irreversible. 3-deoxy-7phosphoheptulonate synthase is activated by its substrates E4P and PEP and is subject of end product
feedback inhibition of trp, tyr and phe.
The subsequent biosynthesis to the end products does not yield any other regulators as for the discussed
chor biosynthesis.
l11.7: 4 ATP + 2 H + 4 NADPH + SO4 + ser => 4 ADP + H2O + 4 NADP + 4 Pi + cys
This reaction results when the sulfate assimilation into PAPS (r11.11), H2S(r11.12), cys (r11.15) and the
reversible acetate assimilation into AcCoA (r7.2) are lumped.
The only irreversible reactions are found in the cys biosynthesis from H2S and are catalyzed by serine Oacetyltransferase and cysteine synthase. Serine O-acetyltransferase is activated by ser and it will be
assumed that it is inhibited by its product of cys. Since the amount of consumed ATP is relative high, also
the regulatory role of the ATP/ADP ratio will be included in the model.
l11.8: 5 ATP + 4 H + 6 NADPH + SO4 + asp => 5 ADP+ H2O + 6 NADP + 5 Pi + homcys
This lumped reaction consists of the sulfate assimilation (r11.11 & r11.12), acetate assimilation (r7.2), the
biosynthesis of homser (r11.18) and the biosynthesis of homcys (r11.13 & r11.14). The reactions r11.11,
r11.12, r7.2 and consists of reversible conversion.
Homcys is synthesized via the intermediate AcHomser. Homeserine-O-acetyltransferase catalyzes the
production of this enzyme and it is inhibited met and the end product homcys. Another inhibitor is cys, but
this is a weak inhibitor and therefore it will be assumed to have a neglectable influence. Subsequently Oacetylhomoserine aminocarboxypropyltransferase catalyzed the conversion of AcHomser into homcys and
is inhibited by met.
Only the first reaction of the homser biosynthesis is irreversible and the involved enzyme, aspartate kinase
is activated by asp and ATP/ADP ratio and is subject of product feedback inhibition by thr.
69
l11.9: 2 ATP+ H2O + H + 2 NADPH + asp => 2 ADP + 2 NADP + 2 Pi + thr
This lumped reaction consists of the homser biosynthesis (r11.18) and thr biosynthesis (r11.19). The
homser biosynthesis has already been discussed for reaction l11.8. The final reaction of the thr synthesis is
irreversible, which is catalyzed by threonine synthase that is being inhibited by cys.
l11.10: 5 ATP+CO2+3 H2O+NADPH+2 NAD+NH4+Rib5P+gln=>5 ADP + 9 H+ 2 NADH + NADP
+ 6 Pi +aKG + his
The reaction has been derived by lumping the PRPP synthesis (r11.31) with the his biosynthesis (r11.32).
This reaction is activated by Rib5P and is inhibited by its product his. Furthermore due to the high energy
consumptions it will be assumed that the ATP/ADP ratio will have a regulatory role.
l11.11: ATP + 2 glu + NADPH => ADP + aKG + orn + H + NADP + Pi
This lumped reaction consists of the ctl biosynthesis (r11.5) except the final intermediate step which
converts orn into ctl. The synthesis of orn is, in chronological order, catalyzed by acetylglutamate kinase,
acetyl-gamma-glutamyl-phosphate reductase, acetylornithine transaminase and finally acetylornithine
deacetylase. The first and the final reaction are irreversible. Acetylglutamate kinase is subject of product
feedback inhibition of arg, but since this amino acid has not been measured, the corresponding elasticity
can not be determined. Furthermore ornithine carbamoyltransferase is activated by glu. Acetylornithine
deacetylase is inhibited by orn.
l11.12: 4 ATP + CO2 + gln + 3 H2O + orn + asp => 4 ADP + glu + 4 H + 4 Pi + arg + fum
This reaction results from the lumping of biosynthesis of carbP (r11.4), the final intermediate reaction of
the biosynthesis of ctl (r11.5), the transport of ctl across the mitochondrial membrane (r10.7) and the
biosynthesis of arg (r11.6).
The synthesis of carbP is catalyzed by carbamoyl-phosphate synthase and is irreversible. The involved
enzyme is subject of end product feedback inhibition of UTP but this effect will be neglected for now to
simplify the model. It will be assumed that gln is a neglectable activator as it is converted into glu, which
in turn is in equilibrium with gln.
70
The biosynthesis of arg from ctl is catalyzed by argininosuccinate synthase and argininosuccinate lyase
which are involved in two sequential reversible reactions.
The conversion of orn into ctl is catalyzed by ornithine carbamoyltransferase, which is irreversible. It is
activated by orn.
Since the amount of energy consumed for the total biosynthesis of arg is relatively high, it will be assumed
that the ATP/ADP will have a regulatory role. No information has been found about the potential product
feedback inhibition of arg despite the fact that most biosynthesis of amino acids are subject of feedback
inhibition of its product. However since arg has not been measured, its corresponding elasticity can not be
determined and therefore will have to be set to zero anyway.
3.3.9 Nucleotide biosynthesis
r13.6: ATP + gln + H2O + UTP => ADP + CTP + glu + 2 H + Pi
This reaction is catalyzed by CTP synthase, which activated by UTP and inhibited by CTP. The influence
of ATP will be neglected.
l13.2: 10 ATP + CO2 + 9 H2O + 2 METHF + 3 NAD + Rib5P + asp + 3 gln + gly => 10 ADP + GMP
+ 17 H + 3 NADH + 10 Pi + 2 THF + fum + 3 glu
This reaction is the result of the lumping of the PRPP synthesis (r11.31), IMP synthesis (r13.1), FTHF
synthesis (r8.3) and the GMP synthesis (r13.3).
The
first
reaction
of
the
IMP
biosynthesis
is
irreversible,
which
is
catalyzed
by
amidophosphoribosyltransferase which is activated by Rib5P and asp and inhibited by AMP.
No effectors have been found for the FTHF synthesis and it is assumed this reaction will be near
equilibrium.
Both intermediate reactions of the GMP synthesis are irreversible. The first reaction is catalyzed by IMP
dehydrogenase which is activated by ATP, CTP, and inhibited by UTP and GMP. The second reaction is
catalyzed by GMP synthase and is activated by ATP.
l13.3: 9 ATP + CO2 + 6 H2O + 2 METHF + 2 NAD + Rib5P + 2 asp + 2 gln + gly => 9 ADP + AMP
+ 15 H + 2 NADH + 9 Pi + 2 THF + 2 fum + 2 glu
71
This lumped reaction consists of the biosynthesis of PRPP (r11.31), IMP (r13.1), FTHF (r8.3) and AMP
(r13.2). The first three syntheses have been discussed in the lumped reaction l13.2 and hence this reaction
is activated by Rib5P and asp and inhibited by AMP.
The first intermediate reaction step of r13.2 is irreversible which is activated by asp and inhibited by
AMP.
l13.4: 6 ATP+2 H2O+NAD+Rib5P+asp+gln=>6 ADP+5 H+NADH+4 Pi+UTP+glu
This reaction consists of the lumped reactions for the IMP synthesis (r13.1) and reversible conversion of
UMP into UTP (r13.5). The IMP synthesis has already been discussed for reaction l13.2. It will be
assumed that the product UTP inhibit the reactions, despite the fact that Brenda does not provide this
information.
3.3.10
ATP hydrolysis
It will be assumed that the elasticities are zero which results that the ATP hydrolysis remains at its steady
state.
3.3.11
Synthesis of glycogen and polysaccharides
r17.7: t6P+H2O => tre + Pi
This reaction is catalyzed trehalose-phosphatase. In the stoichiometric model of van Gulik t6P is only
consumed by the biomass. However Nasution’s glucose response experiment has shown a relatively high
increase in storage material and therefore the t6P consumption have been decoupled from the biomass.
This reaction is activated by t6P.
l17.1: ATP+H2O+2 g6P=>ADP+H+2 Pi+t6P
This reaction consists of 3 intermediate reactions, the reversible synthesis of UDPglc (r17.5), synthesis of
t6P (r17.6) and the reversible conversion of UDP into UTP (r13.9).
The biosynthesis of t6P is irreversible and is catalyzed by trehalose-phosphate synthase. This enzyme is
activated by g6P and is inhibited by UTP and AMP. It is further subject to product feedback inhibition of
t6P. The influence of the ATP/ADP ratio will be neglected
72
l17.4: 0.167 ATP + 0.167 g1P + 0.167 H2O => 0.167 ADP + 0.167 H + 0.333 Pi + psacch
This reaction is activated by g1P and the ATP/ADP ratio. The non-zero elasticity of psacch can not be
determined as psacch has not been measured. Furthermore f16P positively activates the biosynthesis of
psacch.
l17.6: Trehalose part of biomass synthesis
In order to avoid any dead-ends in the stoichiometric model; the incorporation of tre into the biomass has
been modeled in In Silico as a transport across the plasma membrane. It will be assumed that the
elasticityies are set to 0.5
3.3.12
Biomass formation
It will be assumed that the biomass is slightly dependent on the ATP/ADP and that it is saturated with
respect to the amino acids
3.3.13
Penicillin biosynthesis
l19.1: 8 ATP + 4 H2O + O2 + cys + val + PAA => 8 ADP + 8 H + 8 Pi + penG
The penicillin synthesis is the result of the lumping of biosynthesis of ACV (r19.1), iPN (r19.2), penG
(r19.4), PAACoA (r9.3) and the transport across plasma and peroxisomal membrane of PAA and PenG.
The concentrations of the penicillin synthesis intermediates have not measured. Therefore it will be
assumed the reaction is activated by cys, val, aAd and the ATP/ADP ratio. The elasticity of PAA and
PenG will be assumed to be 0.5 as their elasticities can not be determined due to the fact that no
measurements have been made with respect to these metabolites.
3.3.14
Summary
A summary of the chosen regulatory metabolites is shown in Table 3.20. The number of unique non-zero
elasticities is 119. The regulatory function of a metabolite is symbolized by the subscripts + (activator)
73
and – (inhibitor). The subscript * denotes that a corresponding response of a metabolite has not been
measured and furthermore can not be determined from near equilibrium relations.
Table 3.20: Non zero elasticities
#r #ri.i
non-zero elasticities with respect to reactants
1
l1.1
glc+, ATP/ADP ratio+, g6P2
r1.3
f6P+, ATP/ADP ratio+, f16P3
r1.9
PEP+, ADP/ATP ratio+
4
r2.1
g6P+, NADPH5
r2.2
6Pgluct+, NADPH6
r4.1
NADH/NAD-, pyr+, AcCoA/HCoA-, CO2(all identical |ε|)
7
r4.4
iCitr+, NAD/NADH ratio+
8
r4.7
NAD/NADH ratio+, aKG+, succCoA-, CO2-,
(all identical |ε|)
9
r5.1
CO2+, pyr+
10 l6.1
FADH2+, O2+, ATP- (ε linked with stoich
coefficients)
11 l6.4
NADH+, O2+, ATP- (ε linked with stoich
coefficients)
12 r8.1
glu+, THF+, METHF13 r9.2
all ε are set to zero
14 r9.4
all ε are set to zero
15 r9.13 all ε are set to zero
16 r9.14 all ε are set to zero
17 r9.20 all ε are set to zero
18 r11.2 glu+, gln19 r11.3 ATP+, glu+, pro20 r11.7 AcCoA+, aKG+, aAd21 r11.8 aAd+, lys22 r11.9 3PG+, NAD/NADH+, glu+, aKG-, ser- (all
identical |ε|)
23 r11.16 OAA+, asp24 r11.17 asp+, asn25 r11.21 pyr+, ile26 r11.22 pyr+, ala27 r11.34 all ε are set to zero
28 l11.1
pyr+, val29 l11.2
AcCoA+, pyr+, leu30 l11.3
E4P+, PEP+, trp31 l11.4
E4P+, PEP+, tyr32 l11.5
E4P+, PEP+, phe33 l11.7
ser+, ATP/ADP ratio+, cys34 l11.8
asp+, ATP/ADP ratio+,homcys35 l11.9
asp+, thr36 l11.10 ATP/ADP ratio+, Rib5P+, his37 l11.11 glu+, orn-,
38 l11.12 orn+, ATP/ADP+, arg-*
39 r13.6 UTP+, CTP-
effectors
t6P-, citrAMP+, citrf16P+, citrATPATPcitr-, ATP/ADP-
εunique ≠ 0
5
5
4
3
3
3
OAA-
2
2
AcCoA+, asp-
4
1
1
3
0
0
0
0
lys-
2
3
4
2
1
-
2
3
val
ileval-, ile-
met-, thrcysarg-*
0
3
5
3
3
3
3
5
3
3
2
2
2
74
40
l13.2
ATP/ADP ratio+, Rib5P+, asp+, GMP-
AMP-, UTP-,
CTP-
41 l13.3
Rib5P+, asp+, AMP42 l13.4
Rib5P+, asp+, UTP43 r15.1 all ε are set to zero
44 r17.7 t6P+
45 l17.1
g6P+, t6PAMP-, UTP+
+
f16P+
46 l17.4
g1P , ATP/ADP ratio , psacch
+
47 l17.6
tre
48 l18.1
ATP/ADP ratio+
49 l19.1
cys+, val+, aAd+, ATP/ADP+, PenG*, PAA+*
* can not be determined as concentration and response are not known.
7
3
3
0
1
4
4
1
1
5
However since the model has been reduced several of these metabolites have been lumped or elimated
from the stoichiometric model. For example ATP is incorparated in the equilibrium pool p3. Instead of
entering a non-zero elasticity for ATP in the elasticity matrix, a non-zero elasticity for p3 is entered.
Now that the non-zero entries in the elasticity matrix have been specified, the reduced system can
be simulated
4 Discussion
The aim of the project was to develop a dynamic mathematical model for the model organism Penicillium
chrysogenum using approximated linlog kinetics. However this goal has not been achieved due to time
contraints which resulted that no simulation or kinetic parameter estimation has been done.
The first step of developing a kinectic model has been the definition of the stoichiometric model for
Penicillium chrysogenum. The stoichiometric model has been implemented with a new modelling tool In
Silico which required the construction of a database that includes both reactions and transport steps, see
appendix F. Once this database has been constructed, the reactions and transport can be entered and the
metabolic network can be examined for conserved moieties, dead ends, parrellel routes etc. The metabolic
fluxes reported by van Gulik et al. (2000) have been reproduced using both Matlab and In Silico. After a
familarization period, it has been decided to use the In Silico program due to the fact that it visualizes the
system better, allowing a better focus.
Subsequently a literature survey and database base search has been done, see appendix C, in order to
determine the number of enzyme-metabolite interactions and the reversibility of certain reactions. This
requires the identification of all enzymatic reactions in the stoichiometric model and due to the size of the
network; this consumed much more time than has been anticipated. Subsequently the reactor kinectics are
postulated on the basis of full mass action for the stoichiometric model penG. Since the stoichiometric
75
model contains 188 metabolites and 167 reactions, the elasticity matrix for this metabolic reaction network
is 31299 of which based on the literature survey approx 1000 elasticities are identified to be potentially
non-zero. Due to the large number of elasticities, it is expected that the quality of the estimation of the
kinectic parameters is poor considering the amount of data available. Hence it has been decided to reduce
the stoichiometric model by a data-drive approach, which has been described in chapter 3.2.1. This
allowed that several metabolites were lumped away, which reduced the size of the elasticity matrix and
several non-zero entries.
The model has been adjusted for the data provided by Uly Nasution and data reconciliation has been
carried out under the constraint of the elementary conservation relations according to the methodology
decribed by van der Heijden et al. (1994). Later on, this has also been done with In Silico, but elemental
balancing in this program requires the definition of the elementary composition of the metabolites. Since
not all metabolites have been predefined, the elementary balancing has only been performed for the last
metabolic reaction network penGv4. This revealed that several reactions had a proton imbalance which is
probably caused by the fact that some metabolites are defined differently in In Silico than the definition by
van Gulik. Since In Silico is used, the reactions have been redefined as given in appendix B for penGv4.
Subsequently the reversibility of the reactions has been examined, in order to make a selection of potential
reactions that can be assumed to be close to equilibrium. Time scale analysis has been employed to further
reduce the stoichiometric model. In order to make a simulation it is necessary to eliminate the pseudo
equilibrium rates form the model which can be done by lumping the involved metabolites into an
equilibrium pool. Although this might be simple for a relative small and straightforward pathway, to
become increasingly disorderly in highly connected networks like the one discussed in this work. The
methology presented in Visser et al. (2000) offers a systematical approach for subsequently elimating pss
metabolites, PEQ rates and conserved moieties from the model. However in work the order is the
opposite. First the system is checked for dependencies such as conserved moieties which can easily be
removed due to the conservation principle. The second step is to identify the pseudo equilibrium reactions
since it might be possible to calculate the concentration profile. Subsequently the pss steady state
metabolites are identified on the basis on the turnover time or by the fact that they have not been measured
and can not be determined on the basis of the peq assumption. Finally frozen metabolites are identified.
Another dissimilarity between the methology presented in Visser et al. (2000) and in this work is the use
of the null space on a rational basis, instead of calculation the orthonormal basis of the nullspace and the
transformation matrix. This reduced the number of numerical operations. Unfortunately the method has
been employed at a late stage of this project and hence much time has been lost by trying to manual lump
peq metabolites in order to elimate the rates. The manual reducing of the metabolic network could have
76
been done more systematically if time scale analysis has been employed by treating the metabolites that
have been elimated as pss metabolites. However the result should be similar.
The stoichiometric model has been successfully reduced from 188x167 reactions to 41x46 after
elimination of conserved moieties, PEQ rates, pss metabolites and frozen pools.
Although the transient behaviour of metabolites involved in a PEQ rate can be calculated on the basis of
the mass action rate, caution should be taken in calculating the steady state concentrations of these
metabolites, since these require the knowledge of equilibrium constants and often also assumptions
regarding metabolites such as Pi and NH4. Hence the calculated steady state concentrations are more an
indication at what region the concentration may lie. Furthermore the equilibrium constants for some
enzymes in literature seem to vary significantly. For example, Shevchenko (Shevchenko and Guly 1973)
reports the equilibrium constant for isocitrate dehydrogenase of 0.071 whereas Veech (Veech 1968)
reports a significant higher value of 0.91.
In order to calculated the steady state concentrations of the conversed moieties such as NAD or HCoA, the
conservation sum, written as ∑, has been assumed to be equal to 1 mM and furthermore assumed to
remain constant. This can result that the calculated steady state concentrations are wrong although the
concentration of 1 mM is in the region of the other metabolite concentrations. However the assumption
that ∑ does not always hold as can be seen from Figure 4.1, which shows the total sum of the adenosine
species (Nasution et al. 2006). As can be seen the sum of the species does not remain constant under
dynamic conditions. However it will be assumed that the conservation principle also holds under dynamic
conc
umol/gDW
condition, in order to calculate the concentration of the individual conserved moieties.
10
9
8
7
6
5
4
3
2
1
0
ATP
ADP
AMP
cAMP*
-50
-40
-30
-10
25
55
75
110
145
205
265
330
420
time [sec]
Figure 4.1: Adenosine conserved moieties
Apart from that the made assumptions might not be spot on, the transient behaviour of certain
metabolites can be calculated on the basis of two different PEQ reactions. For example
NADH/NAD can be also be calculated by the equilibrium pool between mal, glu, aKG and asp
77
[ mal ]mit ⋅ [Glu ]mit
⎛ NADH ⎞
'
⎜
⎟ =K ⋅
[ aKG ]mit ⋅ [ Asp ]mit
⎝ NAD ⎠ mit
(4.78)
The result of the equation above and the original result of the NADH/NAD is shown in Figure 1.1. As can
be seen the transient behaviour calculated by equation (4.78) follows the orginal result with some delay.
6
5
norm
4
3
2
norm (NADH/NAD):cyt
norm (NADH/NAD):mit
1
-100
0
100
sec
200
300
400
500
Figuur 4.1: Normalized NADH/NAD ratio
It illustrates the fact that not only the assumptions with respect to the NH4, Pi and ∑ are important but also
the choice of the pseudo equilibrium reaction. In this case the preference lies in the peq rate with the
highest metabolic flux, as the influence on the other involved metabolites is much more significant than
for reaction with relatively small rate.
Nevertheless the main Achilles heel of the proposed model is the neglectance of the mitochondrial
compartment, which might result that the energy housekeeping of the cell is wrongly described, as already
discussed in chapter 2.6.1. An option to incorporate this effect is to make a mathematically destinction
between cytosol and mitochondrial NADH on the basis of a concentration ratio between the
compartments. Theobald has used the following value for this concentration ratio (Theobald et al. 1997):
⎛ NAD : cyt ⎞
⎛ NAD : mit ⎞
⎜ NADH : cyt ⎟ = 100 ⋅ ⎜ NADH : mit ⎟
⎝
⎠
⎝
⎠
(4.79)
However this does not change the fact that the NAD/NADH ratio is still calculated on the basis of the
average ATP/ADP concentration. However again due to the time constraints this has not be done.
5 Conclusion
In this work, the development of a large scale kinectic model of a metabolic network for Penicillium
chrysogenum is elaborated. The kinectic model will be based on the stoichiometric model reported in van
Gulik et al. (2000), which contains 188 metabolites and 167 reactions. This model has been reduced to 41
metabolites and 46 dynamic reactions by removing the conserved moeiteies, elimination of PEQ rates and
78
pss metabolites and frozen pools. Subsequentlty the reaction kinectis has been postulated on the basis of a
literature survey and online database search. Due to time constraint no simulation has be done.
6 Nomenclature
Abbreviation Full name
13PG
1,3-bisphosphoglycerate
2PG
2-phosphoglycerate
3PG
3-phosphoglycerate
6APA
6-aminopenicillinic acid
6Pgluct 6-phosphogluconate
8HPA
8-hydroxypenillic acid
A
adenosine
aAd
a-aminoadipate
Aald
acetaldehyde
AApool pool of free amino acids
AAprotsyn amino acids for protein synthesis
Ac
acetate
Accarn
acetylcarnitine
AcCoA acetyl coenzyme A
AcHomser o-acetylhomoserine
ACV
d-(a-aminoadipyl)-cysteinylvaline
ADP
adenosine-5-diphosphate
aKB
a-ketobutyrate
aKG
a-ketoglutarate
aKI
a-ketoisovalerate
ala
alanine
AMP
adenosine-5-monophosphate
arg
arginine
asn
asparagine
asp
aspartate
ATP
adenosine-5-triphosphate
bIM
b-isopropylmalate
biom_cc_gluc average biomass composition of glucose-limited cultures
biom_cc_etoh average biomass composition of ethanol-limited cultures
biom_cc_acet average biomass composition of acetate-limited cultures
carbP
carbamoylphosphate
carn
carnitine
CDPDAcgcl cytidine diphosphate-diacylglyerol
chit
chitine
chor
chorismate
citr
citrate
CMP
cytidine monophosphate
CO2
carbon dioxide
ctl
citruline
CTP
cytidine triphosphate
CYON
cystathionine
cys
cysteine
DHAP
dihydroxyacetone phosphate
E4P
erythrose-4-phosphate
ergo
ergosterol
ery
erythritol
ESE
ergosterolester
EtOH
ethanol
ExPept
excreted peptides
f16P
fructose-1,6-bisphosphate
f6P
fructose-6-phosphate
FAD
flavine adenine dinucleotide (oxidized)
FADH2 flavine adenine dinucleotide (reduced)
FTHF
formyltetrahydrofolate
fum
fumarate
g6P
glucose-6-phosphate
GAP
3-phosphoglyceraldehyde
79
gcl
gcl3P
glc
gln
glu
gluct
gly
glyox
GMP
H
H2O
H2S
HCoA
his
homcys
homser
iCitr
ile
IMP
ino
iPN
lano
leu
linCoA
lys
m1P
mal
man
met
METHF
meva
MYTHF
NAD
NADH
NADP
NADPH
NH4
NO2
NO3
O2
OAA
OHPAA
olCoA
olLinGcl
OPC
PAA
PAACoA
PAPS
penG
PEP
PHchol
phe
PHeta
PHino ph
phospht
PHser
Pi o
pre
pro
PROT
PRPP
psacch
pyr
Rib5P
Ribu5P
RNA
SAH
SAM
sed7P
ser
glycerol
3-phosphoglycerol
glucose
glutamine
glutamate
gluconate
glycine
glyoxylate
guanosine monophosphate
proton
water
hydrogen sulfide
coenzyme A
histidine
homocysteine
homoserine
isocitrate
isoleucine
inosine monophosphate
inositol
isopenicilline
lanosterol
leucine
linoleoyl coenzyme A
lysine
mannitol-1-phosphate
malate
mannitol
methionine
methylene tetrahydrofolate
mevalonate
methyltetrahydrofolate
nicotinamide adenine dinucleotide (oxidized)
nicotinamide adenine dinucleotide (reduced)
nicotinamide adenine dinucleotide phosphate
nicotinamide adenine dinucleotide phosphate
ammonia
nitrite
nitrate
oxygen
oxaloacetate
orthohydroxyphenylacetic acid
oleoyl coenzyme A
oleoyllinoleoyl glycerol
6-oxopiperidine-2-carboxylic acid
phenylacetic acid
phenylacetyl coenzyme A
3-phosphoadenosine-5-phosphosulfate
penicillin-G
phosphoenolpyruvate
phosphatidylcholine
phenylalanine
phosphatidylethanolamine
osphatidylinositol
phosphatidate
phosphatidylserine
rthophosphate
prephenate
proline
protein
a-5-phosphoribosylpyrophosphate
polysaccharides
pyruvate
ribose-5-phosphate
ribulose-5-phosphate
ribose nucleic acid
S-adenosylhomocysteine
S-adenosylmethionine
sedoheptulose-7-phosphate
serine
80
SO4
sulfate
steaCoA stearoyl coenzyme A
succ succinate
succCoA succinyl coenzyme A
t6P
trehalose-6-phosphate
THF
tetrahydrofolate
thr
threonine
tre
trehalose
TRIA
triacylglycerol
trp
tryptophane
tyr
tyrosine
UDP
uridine-5-diphosphate
UDPglc uridine-5-diphosphoglucose
UMP
uridine monophosphate
UTP
uridine triphosphate
val
valine
xol
xylitol
xyl
xylose
xylu
xylulose
Xylu5P xylulose-5-phosphate
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83
Appendix A: Net Conversion Rates
The time dependent perturbation data provided by Uly Nasution (Nasution et al. 2006) is used to estimate
the kinetic parameters. In Table A. 1 the available perturbation data is shown from different glucose
response experiments. As can be seen the experimental conditions of these three experiments are very
similar.
Table A. 1: Experimental details
Experiment
Cglc
D
Cx
-1
CC137
0.5 g/l
0.05 h
CC139
0.23 g/l
0.05 h-1
0.25 g/l
-1
Perturbation and fermentation data available
6 gDw/l
M(11), AA(0), N(3), fermentation data, off gas
6 gDW/l
M(16), AA(0), N(10), glc, fermentation data, off
gas
CC157
0.06 h
6 gDW/l
M(19), AA (22), N(9), glc, no fermentation data
Abbreviationsa: Cglc = glucose concentration at pulse, D= dilution rate, Cx = biomass concentration, M = perturbation data metabolites central
pathways, AA = perturbation data amino acids, N = perturbation data nucleotides, glc = perturbation data glucose.
Experiment CC137 provides the perturbation data of 11 metabolites from central pathways and 3
nucleotides, off gas measurements of O2 and CO2 and the steady state measurements of the fermentor. The
glucose concentration in experiment CC137 is twice as high compared to the other experiments, but it has
been shown that the mass uptake of glucose already reached its maximum value of 42 mmol/Cmol hr at a
concentration of 0.25 g/l (Nasution 2007). However balancing the net conversion rates revealed that there
is an error in measurements or wrong system definition with a confidence level of 97% based on the
biomass composition of CH1.865N0.159O0.627P0.0246S0.01-0.014. Using the biomass composition provided by van
Gulik, see chapter 2.1, resulted in a confidence level of 98%.
Balancing the net conversion rates of experiment CC139 resulted that there is no proof of an error in
measurements or wrong system definition as the error significance is 15% based on the biomass
composition provided by van Gulik. Hence the steady state measurements and off gas measurements of
experiment CC139 are used.
Experiment CC157 provides the most time dependent perturbation data and will therefore be used to
estimate the kinectic parameters.
In Table A. 2 the steady state measurements of experiment CC139 has been listed, which are used to
calculate the net conversion rates, see chapter 3.1.
Table A. 2: Steady State measurements fermentor of experiment CC139
-1-
Measurement
Unit
Variable
Value
Variance
name
mmol/Cmol/hr
effluent flow
L/h
φuit
1.990E-01
3.962E-06
medium feed flow
L/h
φin
1.969E-01
0.000E+00
L
V
4.000E+00
6.400E-03
φgas
9.334E+00
3.485E-02
working volume
gasflow in
mol/h
oxygen in
%
XO2,g,feed
2.095E+01
6.280E-28
oxygen out
%
XO2,g,out
2.044E+01
6.753E-03
carbon dioxide in
%
XCO2,g,feed
1.329E-02
3.285E-04
carbon dioxide out
%
XCO2,g,out
4.844E-01
6.071E-04
gDw/L
Cx
5.988E+00
6.981E-03
gDw/Cmol
Mw
2.805E+01
3.147E-01
biomass concentration
Cmol weight
glucose feed concentration
mmol/L
Xglc,feed
8.333E+01
2.778E+00
ammonium feed concentration
mmol/L
Xnh4,feed
0.000E+00
0.000E+00
PAA feed concentration
mmol/L
Xpaa,feed
4.850E+00
9.409E-03
glucose residual concentration
mmol/L
Xglc
1.491E-04
3.714E-11
ammonium residual concentration
mmol/L
Xnh4
N/A
PAA residual concentration
mmol/L
Xpaa
2.993E+00
7.020E-02
Pen-G concentration
mmol/L
Xpeng
1.402E+00
4.065E-02
PIO concentration
mmol/L
Xpio
N/A
N/A
IPN concentration
mmol/L
Xipn
N/A
N/A
6-APA concentration
mmol/L
X6apa
N/A
N/A
8-HPA concentration
mmol/L
X8hpa
N/A
N/A
OH-PAA concentration
mmol/L
Xohpaa
N/A
N/A
OPC concentration
mmol/L
Xopc
N/A
N/A
mCmol/L
Xaa
N/A
N/A
total organic carbon concentration
mCmol/L
Xtoc
9.806E+01
Cell volume
ml/gDW
Vcell
2.3
amino acids and peptides
N/A
concentration
2.389E+01
-2-
f6P
5000
4000
3000
2000
CC137
1000
CC139
0
CC157
-55
45
145
( se c ) 245
345
445
900
800
70 0
600
50 0
400
300
200
10 0
0
-55
g1P
800
600
(umol/l)
g6P
400
200
0
-55
14 5
(s ec)
f16P
2PG + 3PG
(sec)
345
PEP
3000
300
145
345
150
200
2000
150 0
100
(umol/l)
500
400
300
200
100
0
-55
0
-55
50 0
145
(sec)
345
0
-55
14 5
(s ec)
2000
150 0
10 0 0
50 0
(sec)
345
0
-55
14 5
(s ec)
345
18 0 0
16 0 0
14 0 0
12 0 0
10 0 0
800
600
400
200
0
-55
345
200
4000
150
3000
500
10 0
2000
50
10 0 0
(sec)
345
0
-55
(umol/l)
1500
1000
500
(sec)
(s ec)
345
-55
14 5
345
500
400
300
200
100
0
-55
(s ec)
345
f26P
succ
2000
145
0
14 5
8
(umol/l)
145
t6P
0
-55
(s ec)
2 50
50 0 0
1000
345
6Pgluc
6000
1500
0
-55
14 5
mal
fum
(sec)
aKG
2 50 0
145
145
345
citr+iC itr
pyr
(umol/l)
50
10 0 0
0
-55
(umol/l)
100
(umol/l)
(umol/l)
2 50 0
145
(sec)
345
6
4
2
0
-55
145
(sec)
345
Figure 0.1: Transient behaviour of metabolites of main pathways after glucose pulse
-3-
ATP
ADP
CC137
1000
0
-55
CC139
145
(sec)
345CC157
1000
800
600
400
200
0
-55
145
(sec)
145
(sec)
300
200
100
0
-55
345
345
500
400
300
200
100
0
-55
145
(sec)
145
(sec)
345
GMP
CTP
(umol/l)
(umol/l)
UDP
400
(umol/l)
2000
1000
800
600
400
200
0
-55
(umol/l)
3000
(umol/l)
(umol/l)
4000
AMP
345
50
40
30
20
10
0
-55
145
(sec)
345
Figure 0.2: Transient behaviour of nucleotides after glucose pulse
Figure 0.3: Transient behaviour of amino acids after glucose pulse
-4-
Appendix B: Lumped Reactions
In Table B. 1 the metabolic reaction network is presented of the penG model, which consists of 167
reactions located in the cytosol, mitochondria and peroxisome. The symbol J0van Gulik denotes the metabolic
fluxes which have been calculated by van Gulik (van Gulik et al. 2000) for a glucose-limited strain of
Penicillium chrysogenum at a dilution rate of 0.3 hr-1. The symbol J0 denotes the steady state metabolic
fluxes calculated by the In Silico model penGv1. As can be seen from the table the results have been
reproduced.
Table B. 1: Metabolic Reaction Network of penG model
ri.i
Reaction
location
J0
J0van Gulik
Cmol. hr
Cmol. hr
mmol
mmol
Glycolysis
r1.1
ATP + glc => ADP + g6P + H
r1.2
g6P => f6P
r1.3
ATP + f6P => ADP + f16P + H
r1.4
f16P => 2 GAP
r1.5
GAP + NAD + Pi => 13PG + H + NADH
r1.6
13PG + ADP => 3PG + ATP
r1.7
3PG => 2PG
r1.8
2PG => H2O + PEP
r1.9
ADP + H + PEP => ATP + pyr
PPP
r2.1
g6P + H2O + NADP => 6Pgluct + 2 H + NADPH
r2.2
6Pgluct + NADP => CO2 + NADPH + Ribu5P
r2.3
Ribu5P => Rib5P
r2.4
Ribu5P => Xylu5P
r2.5
Rib5P + Xylu5P => GAP + sed7P
r2.6
GAP + sed7P => E4P + f6P
r2.7
E4P + Xylu5P => f6P + GAP
TCA cycle
r4.1
HCoA + NAD + pyr => AcCoA + CO2 + NADH
r4.2
AcCoA + H2O + OAA => citr + H + HCoA
r4.3
citr => iCitr
r4.4
iCitr + NAD => aKG + CO2 + NADH
r4.5
iCitr + NADP => aKG + CO2 + NADPH
r4.6
iCitr + NADP => aKG + CO2 + NADPH
r4.7
aKG + HCoA + NAD => CO2 + NADH + succCoA
r4.8
ADP + Pi + succCoA => ATP + HCoA + succ
r4.9
FAD + succ => FADH2 + fum
r4.10
fum + H2O => mal
r4.11
mal + NAD => H + NADH + OAA
Anapleoritc Pathway
cyt
cyt
cyt
cyt
cyt
cyt
cyt
cyt
cyt
13.06
6.03
8.82
8.82
19.04
19.04
17.76
17.76
17.29
13.06
6.03
8.82
8.82
19.04
19.04
17.76
17.76
17.29
cyt
cyt
cyt
cyt
cyt
cyt
cyt
5.10
5.10
1.69
3.12
1.69
1.69
1.43
5.10
5.10
1.69
3.12
1.69
1.69
1.43
mit
mit
mit
mit
mit
cyt
mit
mit
mit
mit
mit
12.85
12.63
10.59
6.90
1.43
2.26
9.53
9.53
9.53
9.89
12.63
12.85
12.63
10.59
6.90
1.43
2.26
9.53
9.53
9.53
9.89
12.63
-5-
r5.1
ATP + CO2 + H2O + pyr => ADP + 2 H + OAA + Pi
cyt
r5.2
ATP + citr + HCoA => AcCoA + ADP + OAA + Pi
cyt
r5.3
H + NADH + OAA => mal + NAD
cyt
Oxidative Phosphorylation
r6.1
11 H:mit + NADH:mit + 0.5 O2:cyt => 10 H:cyt + H2O:cyt + NAD:mit
r6.2
7 H:mit + NADH:cyt + 0.5 O2:cyt => 6 H:cyt + H2O:cyt + NAD:cyt
r6.3
FADH2:mit + 6 H:mit + 0.5 O2:cyt => FAD:mit + 6 H:cyt + H2O:cyt
r6.4
ADP:mit + 4 H:cyt + Pi:mit => ATP:mit + 3 H:mit + H2O:mit
Different Carbon Substrates
r7.2
Ac + 2 ATP + H2O + HCoA => AcCoA + 2 ADP + H + 2 Pi
cyt
Transfer of 1-C Compounds
r8.1
gly + NAD + THF => CO2 + METHF + NADH + NH4
cyt
r8.2
H + METHF + NADH => MYTHF + NAD
cyt
r8.3
ATP + 2 H2O + METHF + NAD => ADP + FTHF + 2 H +
cyt
NADH + Pi
r8.4
ATP + 2 H2O + met => H + 3 Pi + SAM
cyt
r8.5
H2O + SAH => A + homcys
cyt
Transport Across the Plasma Membrane
R9.1
ATP:cyt + H2O:cyt => ADP:cyt + H_external + Pi:cyt
R9.2
2 H_external + Pi_external => 2 H:cyt + Pi:cyt
R9.3
glc_external + H_external => glc:cyt + H:cyt
R9.4
2 H_external + SO4_external => 2 H:cyt + SO4:cyt
R9.6
O2_external => O2:cyt
R9.7
CO2:cyt => CO2_external
R9.8
penG:cyt => penG_external
R9.9
6APA:cyt => 6APA_external
R9.10
8HPA:cyt => 8HPA_external
R9.11
PAA_external => PAA:cyt
R9.12
OHPAA:cyt => OHPAA_external
R9.13
ExPept:cyt => ExPept_external
R9.14
psacch:cyt => psacch_external
R9.15
H2O:cyt => H2O_external
R9.16
OPC:cyt => OPC_external
R9.17
iPN:cyt => iPN_external
R9.20
H_external + NH4_external => H:cyt + NH4:cyt
R9.23
PIO:cyt => PIO_external
Transport Across the Mitochondrial Membrane
R10.1
ADP:cyt + ATP:mit => ADP:mit + ATP:cyt
R10.3
H2O:mit => H2O:cyt
R10.5
H:cyt + Pi:cyt => H:mit + Pi:mit
R10.6
citr:cyt + iCitr:mit => citr:mit + iCitr:cyt
R10.7
ctl:mit + H:cyt => ctl:cyt + H:mit
R10.8
mal:mit + Pi:cyt => mal:cyt + Pi:mit
R10.9
NH4:mit => NH4:cyt
R10.10
H:cyt + ile:mit => H:mit + ile:cyt
R10.11
H:cyt + pyr:cyt => H:mit + pyr:mit
R10.12
CO2:mit => CO2:cyt
R10.13
fum:cyt + H:cyt => fum:mit + H:mit
R10.14
citr:mit + mal:cyt => citr:cyt + mal:mit
1.74
2.03
2.74
1.74
2.03
2.74
41.92
18.35
9.53
149.28
41.92
18.35
9.53
149.28
0.70
0.70
-0.05
0.09
0.23
-0.05
0.09
0.23
0.08
0.08
0.08
0.08
27.07
0.25
13.06
0.70
35.79
38.05
0.58
0.04
0.03
0.63
0.04
1.15
2.59
55.17
0.18
0.00
6.79
0.00
27.07
0.25
13.06
0.70
35.79
38.05
0.58
0.04
0.03
0.63
0.04
1.15
2.59
55.17
0.18
0.00
6.79
0.00
158.28
127.44
156.73
2.26
0.18
1.56
0.14
0.14
15.22
31.79
0.36
4.30
158.28
127.44
156.73
2.26
0.18
1.56
0.14
0.14
15.22
31.79
0.36
4.30
-6-
R10.15
H:cyt + thr:cyt => H:mit + thr:mit
R10.16
glu:cyt + H:cyt => glu:mit + H:mit
R10.17
H:cyt + val:mit => H:mit + val:cyt
R10.18
gln:cyt + H:cyt => gln:mit + H:mit
R10.19
bIM:mit + H:cyt => bIM:cyt + H:mit
Amino Acid synthesis
r11.1
aKG + H + NADPH + NH4 => glu + H2O + NADP
r11.2
ATP + glu + NH4 => ADP + gln + H + Pi
r11.3
ATP + glu + 2 H + 2 NADPH => ADP + H2O + 2 NADP +
Pi + pro
r11.4
2 ATP + CO2 + gln + 2 H2O => 2 ADP + carbP + glu + 3 H
+ Pi
r11.5
ATP + carbP + 2 glu + NADPH => ADP + aKG + ctl + H +
NADP + 2 Pi
r11.6
asp + 2 ATP + ctl + H2O => 2 ADP + arg + fum + H + 2 Pi
r11.7
AcCoA + glu + H2O + NAD => aAd + CO2 + HCoA +
NADH
r11.8
aAd + 2 ATP + glu + H2O + NAD + 2 NADPH => 2 ADP +
aKG + H + lys + NADH + 2 NADP + 2 Pi
r11.9
3PG + glu + H2O + NAD => aKG + H + NADH + Pi + ser
r11.10
ser + THF => gly + H2O + METHF
r11.11
2 ATP + 3 H + H2O + SO4 => ADP + PAPS + 2 Pi
r11.12
H + 4 NADPH + PAPS => ADP + 3 H2O + H2S + 4 NADP
r11.13
AcCoA + homser => AcHomser + HCoA
r11.14
AcHomser + H2S => Ac + H + homcys
r11.15
AcCoA + H2S + ser => Ac + cys + H + HCoA
r11.16
glu + OAA => aKG + asp
r11.17
asp + 2 ATP + H2O + NH4 => 2 ADP + asn + 2 H + 2 Pi
r11.18
asp + ATP + 2 H + 2 NADPH => ADP + homser + 2 NADP
+ Pi
r11.19
ATP + H2O + homser => ADP + H + Pi + thr
r11.20
homcys + MYTHF => met + THF
r11.21
glu + 2 H + NADPH + pyr + thr => aKG + CO2 + H2O + ile
+ NADP + NH4
r11.22
glu + pyr => aKG + ala
r11.23
2 H + NADPH + 2 pyr => aKI + CO2 + H2O + NADP
r11.24
aKI + glu => aKG + val
r11.25
AcCoA + aKI + H2O => bIM + H + HCoA
r11.26
bIM + glu + NAD => aKG + CO2 + leu + NADH
r11.27
ATP + E4P + NADPH + 2 PEP => ADP + chor + NADP + 4
Pi
r11.28
chor + glu + H => aKG + CO2 + H2O + phe
r11.29
chor + glu + NAD => aKG + CO2 + NADH + tyr
r11.30
chor + gln + PRPP + ser => CO2 + GAP + glu + H + H2O +
2 Pi + pyr + trp
r11.31
2 ATP + Ribu5P => 2 ADP + H + PRPP
r11.32
3 ATP + CO2 + gln + 3 H2O + 2 NAD + NADPH + NH4 +
PRPP => 3 ADP + aKG + 8 H + his + 2 NADH + NADP + 6
Pi
r11.33
0.137 ala + 0.0273 arg + 0.0358 asn + 0.061 asp + 0.00526
cys + 0.175 gln + 0.267 glu + 0.0336 gly + 0.0294 his +
0.0137 ile + 0.0168 leu + 0.0231 lys + 0.00526 met + 0.00526
0.14
1.20
0.89
0.18
0.23
0.14
1.20
0.89
0.18
0.23
cyt
cyt
cyt
5.59
1.10
0.17
5.59
1.10
0.17
mit
0.18
0.18
mit
0.18
0.18
cyt
cyt
0.18
0.32
0.18
0.32
cyt
0.14
0.14
cyt
cyt
cyt
cyt
cyt
cyt
cyt
cyt
cyt
cyt
1.28
0.37
0.70
0.70
0.04
0.04
0.66
1.03
0.12
0.36
1.28
0.37
0.70
0.70
0.04
0.04
0.66
1.03
0.12
0.36
cyt
cyt
mit
0.32
0.12
0.14
0.32
0.12
0.14
cyt
mit
mit
mit
cyt
cyt
0.36
1.11
0.89
0.23
0.23
0.23
0.36
1.11
0.89
0.23
0.23
0.23
cyt
cyt
cyt
0.13
0.07
0.03
0.13
0.07
0.03
cyt
cyt
0.29
0.08
0.29
0.08
cyt
0.23
0.23
-7-
phe + 0.0578 pro + 0.0494 ser + 0.0326 thr + 0.0021 trp +
0.00631 tyr + 0.0168 val => 4.56 AApool
r11.34
0.134 ala + 0.07 arg + 0.0386 asn + 0.0386 asp + 0.02 cys +
0.0509 gln + 0.0509 glu + 0.132 gly + 0.0119 his + 0.0307 ile
+ 0.0559 leu + 0.0348 lys + 0.0103 met + 0.0266 phe +
0.0537 pro + 0.044 ser + 0.0467 thr + 0.01 trp + 0.0203 tyr +
0.12 val => 4.53 ExPept + H2O
Protein Synthesis
r12.1
0.113 ala + 0.0572 arg + 0.038 asn + 0.038 asp + 0.00459 cys
+ 0.0503 gln + 0.0503 glu + 0.102 gly + 0.025 his + 0.0486
ile + 0.0784 leu + 0.0485 lys + 0.0133 met + 0.0478 phe +
0.0554 pro + 0.0561 ser + 0.0608 thr + 0.0099 trp + 0.0242
tyr + 0.0785 val => AAprotsyn
r12.2
AAprotsyn + 4 ATP + 3 H2O => 4 ADP + 4 H + 4 Pi + 4.81
PROT
Nucleotide Synthesis
r13.1
asp + 4 ATP + CO2 + 2 FTHF + 2 gln + gly + 2 H2O + PRPP
=> 4 ADP + fum + 2 glu + 8 H + IMP + 6 Pi + 2 THF
r13.2
asp + ATP + IMP => ADP + AMP + fum + 2 H + Pi
r13.3
2 ATP + gln + 3 H2O + IMP + NAD => 2 ADP + glu + GMP
+ 4 H + NADH + 2 Pi
r13.4
asp + 2 ATP + gln + 2 H2O + NAD + PRPP => 2 ADP + glu
+ 4 H + NADH + 4 Pi + UMP
r13.5
2 ATP + UMP => 2 ADP + UTP
r13.6
ATP + gln + H2O + UTP => ADP + CTP + glu + 2 H + Pi
r13.7
2 ADP + CTP => 2 ATP + CMP
r13.8
A + 2 ATP => 3 ADP + H
r13.9
ATP + UDP => ADP + UTP
RNA Synthesis
r14.1
0.349 AMP + 3.23 ATP + 0.168 CMP + 0.26 GMP + 2.23
H2O + 0.222 UMP => 3.23 ADP + 3.23 H + 3.23 Pi + 9.61
RNA
ATP Hydrolysis
r15.1
ATP + H2O => ADP + H + Pi
Synthesis of Fatty Acids
r16.1
g6P + H2O => ino + Pi
r16.2
GAP + H + NADH => gcl3P + NAD
r16.3
9 AcCoA + 8 ATP + 8 H + 16 NADPH => 8 ADP + 8 HCoA
+ 16 NADP + 8 Pi + steaCoA
r16.4
H + NADH + O2 + steaCoA => 2 H2O + NAD + olCoA
r16.5
H + NADH + O2 + olCoA => 2 H2O + linCoA + NAD
r16.6
gcl3P + linCoA + olCoA => 2 HCoA + phospht
r16.7
CTP + H2O + phospht => CDPDAcgcl + 2 Pi
r16.8
CDPDAcgcl + ser => CMP + H + PHser
r16.9
H + PHser => CO2 + PHeta
r16.10
PHeta + 3 SAM => 3 H + PHchol + 3 SAH
r16.11
CDPDAcgcl + ino => CMP + H + PHino
r16.12
H2O + phospht + steaCoA => HCoA + Pi + TRIA
r16.13
3 AcCoA + H + H2O + 2 NADPH => 3 HCoA + meva + 2
NADP
r16.14
18 ATP + 5 H2O + 6 meva + 2 NADPH + O2 => 18 ADP + 6
CO2 + 10 H + lano + 2 NADP + 18 Pi
cyt
0.25
0.25
cyt
2.65
2.65
cyt
2.65
2.65
cyt
0.11
0.11
cyt
cyt
0.07
0.05
0.07
0.05
cyt
0.07
0.07
cyt
cyt
cyt
cyt
cyt
0.03
0.03
-0.02
0.08
0.03
0.03
0.03
-0.02
0.08
0.03
cyt
0.19
0.19
cyt
114.39
114.39
cyt
cyt
cyt
0.01
0.06
0.14
0.01
0.06
0.14
cyt
cyt
cyt
cyt
cyt
cyt
cyt
cyt
cyt
cyt
0.13
0.06
0.06
0.05
0.04
0.04
0.03
0.01
0.01
0.08
0.13
0.06
0.06
0.05
0.04
0.04
0.03
0.01
0.01
0.08
cyt
0.01
0.01
-8-
r16.15
lano + NAD + 2 THF => ergo + H + 2 MYTHF + NADH
cyt
r16.16
ergo + olCoA => ESE + HCoA
cyt
Synthesis of Glycogen and Polysaccharides
r17.1
0.167 ATP + 0.167 g6P + 0.167 H2O => 0.167 ADP + 0.167
cyt
H + 0.333 Pi + 1 psacch
r17.2
AcCoA + f6P + gln => chit + glu + H + HCoA + Pi
cyt
r17.3
f6P + H + NADH => m1P + NAD
cyt
r17.4
H2O + m1P => man + Pi
cyt
r17.5
g6P + H2O + UTP => 2 Pi + UDPglc
cyt
r17.6
g6P + UDPglc => H + t6P + UDP
cyt
r17.7
H2O + t6P => Pi + tre
cyt
r17.8
E4P + H + H2O + NADH => ery + NAD + Pi
cyt
Biomass Formation
r18.17
0.0342 AApool:cyt + 0.00839 chit:cyt + 0.000991 ery:cyt + 0.00043
ESE:cyt + 0.0806 H2O:cyt + 0.00246 man:cyt + 0.000861 PHchol:cyt +
0.000516 PHeta:cyt + 0.000344 PHino:cyt + 0.422 PROT:cyt + 0.282
psacch:cyt + 0.0597 RNA:cyt + 0.00107 tre:cyt + 0.000215 TRIA:cyt =>
biomccgluc_external
Penicillin Biosynthesis
r19.1
aAd + 6 ATP + cys + 4 H2O + val => ACV + 6 ADP + 6 H +
cyt
6 Pi
r19.2
ACV + O2 => 2 H2O + iPN
cyt
r19.3
2 ATP + H2O + HCoA + PAA => 2 ADP + 2 H + PAACoA
per
+ 2 Pi
r19.4
H2O + iPN + PAACoA => aAd + HCoA + penG
per
r19.5
aAd => H2O + OPC
per
r19.6
H + NADPH + O2 + PAA => H2O + NADP + OHPAA
cyt
r19.7
H2O + iPN => 6APA + aAd
per
r19.8
6APA + CO2 => 8HPA
per
r19.9
H2O + penG => PIO
cyt
Transport Across the Peroxisomal Membrane
r21.1
PAA:cyt=>PAA:per
r21.2
iPN:cyt=>iPN:per
r21.3
OPC:per=>OPC:cyt
r21.4
6APA:per=>6APA:cyt
r21.5
8HPA:per=>8HPA:cyt
r21.6
aAd:per=>aAd:cyt
r21.7
penG:per=>penG:cyt
r21.13
ADP:per+ATP:cyt=>ADP:cyt+ATP:per
r21.14
H2O:per=>H2O:cyt
r21.15
H:per+Pi:per=>H:cyt+Pi:cyt
r21.16
CO2:cyt=>CO2:per
0.01
0.01
0.01
0.01
11.10
11.10
0.25
0.07
0.07
0.03
0.03
0.03
0.03
0.25
0.07
0.07
0.03
0.03
0.03
0.03
30.19
30.19
0.64
0.64
0.64
0.58
0.64
0.58
0.58
0.18
0.04
0.06
0.03
0.00
0.58
0.18
0.04
0.06
0.03
0.00
0.58
0.64
0.18
0.04
0.03
0.47
0.58
1.17
-1.05
0.03
1.17
0.58
0.64
0.18
0.04
0.03
0.47
0.58
1.17
-1.05
0.03
1.17
The stoichiometric reaction network of penGv3 is shown in Table B. 2 together with the corresponding
metabolic flux. The metabolic network of the one compartment model penGv4 is shown in Table B. 3.
Table B. 2: Metabolic Reaction Network of penGv3 model
r1.2
Reactions
g6P => f6P
cyt
μmol/gDw/s
0.0842
-9-
r1.3
r1.4
r1.9
l1.1
l1.2
l1.3
r2.1
r2.2
r2.3
r2.4
r2.5
r2.6
r2.7
r4.1
r4.2
r4.3
r4.4
r4.5
r4.6
r4.7
r4.8
r4.9
r4.10
r4.11
r5.1
r5.2
r5.3
l6.1
l6.2
l6.3
r6.4
r8.1
r8.2
R9.1
R9.2
R9.4
R9.6
R9.7
R9.8
R9.11
R9.13
R9.14
R9.15
R9.20
R10.1
R10.10
ATP + f6P => ADP + f16P + H
f16P => 2*GAP
ADP + H + PEP => ATP + pyr
ATP_cyt+ H_external+ glc_external => ADP_cyt + 2*H_cyt + g6P_cyt
GAP+NAD+Pi+ADP = 3PG + ATP + H + NADH
3PG = H2O + PEP
g6P + H2O + NADP => 6Pgluct + 2*H + NADPH
6Pgluct + NADP => CO2 + NADPH + Ribu5P
Ribu5P => Rib5P
Ribu5P => Xylu5P
Rib5P + Xylu5P => GAP + sed7P
GAP + sed7P => E4P + f6P
E4P + Xylu5P => f6P + GAP
HCoA + NAD + pyr => AcCoA + CO2 + NADH
AcCoA + H2O + OAA => citr + H + HCoA
citr => iCitr
iCitr + NAD => aKG + CO2 + NADH
iCitr + NADP => aKG + CO2 + NADPH
iCitr + NADP = aKG + CO2 + NADPH
aKG + HCoA + NAD => CO2 + NADH + succCoA
ADP + Pi + succCoA => ATP + HCoA + succ
FAD + succ => FADH2 + fum
fum + H2O => mal
mal + NAD => H + NADH + OAA
ATP + CO2 + H2O + pyr => ADP + 2*H + OAA + Pi
ATP + citr + HCoA => AcCoA + ADP + OAA + Pi
H + NADH + OAA => mal + NAD
8.36*H_mit + NADH_mit + 0.5*O2_cytosol => 7.36*H_cytosol +
H2O_cytosol + NAD_mit
5.416*H_mit + NADH_cytosol + 0.5*O2_cytosol => 4.416*H_cytosol +
H2O_cytosol + NAD_cytosol
FADH2_mit + 4.416*H_mit + 0.5*O2_cytosol => FAD_mit +
4.416*H_cytosol + H2O_cytosol
ADP_mit + 4*H_cytosol + Pi_mit => ATP_mit + 3*H_mit + H2O_mit
gly + NAD + THF => CO2 + METHF + NADH + NH4
H + METHF + NADH => MYTHF + NAD
ATP_cyt + H2O_cyt => ADP_cyt + H_external + Pi_cyt
2*H_external + Pi_external => 2*H_cyt + Pi_cyt
2*H_external + SO4_external => 2*H_cyt + SO4_cyt
O2_external => O2_cyt
CO2_cyt => CO2_external
penG_cyt => penG_external
PAA_external => PAA_cyt
ExPept_cyt => ExPept_external
psacch_cyt => psacch_external
H2O_cyt => H2O_external
H_external + NH4_external => H_cyt + NH4_cyt
ADP_cyt + ATP_mit => ADP_mit + ATP_cyt
H_cyt + ile_mit => H_mit + ile_cyt
cyt
cyt
cyt
cyt
cyt
cyt
cyt
cyt
cyt
cyt
cyt
cyt
mit
mit
mit
mit
mit
cyt
mit
mit
mit
mit
mit
cyt
cyt
cyt
0.1216
0.1216
0.2392
0.1911
0.2617
0.2475
0.0709
0.0709
0.0282
0.0427
0.0237
0.0237
0.0190
0.1793
0.1753
0.1407
0.0938
0.0175
0.0294
0.1248
0.1248
0.1248
0.1306
0.1753
0.0277
0.0345
0.0447
0.5731
0.2398
0.1248
cyt
cyt
1.4897
-0.0013
0.0017
0.3959
0.0038
0.0043
0.4760
0.5080
0.0032
0.0032
0.0426
0.0767
0.7457
0.0994
1.6048
0.0025
- 10 -
R10.11
R10.12
R10.13
R10.14
R10.15
R10.16
R10.17
R10.3
R10.5
R10.6
R10.8
R10.9
r11.1
r11.10
r11.16
r11.17
r11.2
r11.20
r11.21
r11.22
r11.3
r11.34
r11.7
r11.8
r11.9
r13.6
r15.1
r17.7
l11.1
l11.2
l11.3
l11.4
l11.5
l11.6
l11.7
l11.8
l11.9
H_cyt + pyr_cyt => H_mit + pyr_mit
CO2_mit => CO2_cyt
fum_cyt + H_cyt => fum_mit + H_mit
citr_mit + mal_cyt => citr_cyt + mal_mit
H_cyt + thr_cyt => H_mit + thr_mit
glu_cyt + H_cyt => glu_mit + H_mit
H_cyt + val_mit => H_mit + val_cyt
H2O_mit => H2O_cyt
H_cyt + Pi_cyt => H_mit + Pi_mit
citr_cyt + iCitr_mit => citr_mit + iCitr_cyt
mal_mit + Pi_cyt => mal_cyt + Pi_mit
NH4_mit => NH4_cyt
aKG + H + NADPH + NH4 => glu + H2O + NADP
cyt
ser + THF => gly + H2O + METHF
cyt
glu + OAA => aKG + asp
cyt
asp + 2*ATP + H2O + NH4 => 2*ADP + asn + 2*H + 2*Pi
cyt
ATP + glu + NH4 => ADP + gln + H + Pi
cyt
homcys + MYTHF => met + THF
cyt
glu + 2*H + NADPH + pyr + thr => aKG + CO2 + H2O + ile + NADP + mit
NH4
glu + pyr => aKG + ala
cyt
ATP + glu + 2*H + 2*NADPH => ADP + H2O + 2*NADP + Pi + pro
cyt
cyt
0.134*ala + 0.07*arg + 0.0386*asn + 0.0386*asp + 0.02*cys +
0.0509*gln + 0.0509*glu + 0.132*gly + 0.0119*his + 0.0307*ile +
0.0559*leu + 0.0348*lys + 0.0103*met + 0.0266*phe + 0.0537*pro +
0.044*ser + 0.0467*thr + 0.01*trp + 0.0203*tyr + 0.12*val =>
4.53*ExPept + H2O
AcCoA + glu + H2O + NAD => aAd + CO2 + HCoA + NADH
cyt
aAd + 2*ATP + glu + H2O + NAD + 2*NADPH => 2*ADP + aKG + H
cyt
+ lys + NADH + 2*NADP + 2*Pi
3PG + glu + H2O + NAD => aKG + H + NADH + Pi + ser
cyt
ATP + gln + H2O + UTP => ADP + CTP + glu + 2*H + Pi
cyt
ATP + H2O => ADP + H + Pi
cyt
H2O + t6P => Pi + tre
cyt
2*H + NADPH + glu + 2*pyr => CO2 + H2O + NADP + aKG + val
mit
AcCoA_mit + H_cyt + NADPH_mit + NAD_cyt + glu_cyt + 2*pyr_mit =>
CO2_mit + CO2_cyt + HCoA_mit + NADH_cyt + NADP_mit + aKG_cyt +
leu_cyt
3*ATP+E4P+NADPH+2*PEP+Rib5P+gln+ser=>3*ADP + CO2 + GAP cyt
+ H2O + 2*H + NADP + 6*Pi + glu + pyr +trp
ATP+E4P+NADPH+NAD+2*PEP+glu=>ADP+CO2+NADH+NADP+4 cyt
*Pi+aKG+tyr
ATP+E4P+H+NADPH+2*PEP+glu=>ADP +CO2 + H2O + NADP +
cyt
4*Pi+aKG+phe
2*ATP_cyt+3*ATP_mit+CO2_mit+H2O_cyt+2*H2O_mit+NADPH_mit+asp_
cyt+gln_cyt+glu_mit+H_cyt=>2*ADP_cyt+3*ADP_mit+6*H_mit+NADP_mit
+2*Pi_cyt+3*Pi_mit+aKG_mit+arg_cyt+fum_cyt
4*ATP + 2*H + 4*NADPH + SO4 + ser => 4*ADP + H2O + 4*NADP + cyt
4*Pi + cys
5*ATP + 4*H + 6*NADPH + SO4 + asp => 5*ADP+ H2O + 6*NADP + cyt
5*Pi + homcys
2*ATP+ H2O + H + 2*NADPH + asp => 2*ADP + 2*NADP + 2*Pi +
cyt
0.2054
0.4265
0.0058
0.0640
0.0025
0.0135
0.0078
1.1876
1.5855
0.0294
0.0193
0.0025
0.0793
0.0062
0.0175
0.0021
0.0179
0.0022
0.0025
0.0067
0.0031
0.0094
0.0025
0.0025
0.0142
0.0004
0.9618
0.0005
0.0078
0.0040
0.0005
0.0013
0.0023
0.0032
0.0036
0.0007
0.0057
- 11 -
l11.10
l13.2
l13.3
l13.4
l17.1
l17.3
l17.4
l17.6
l18.2
l19.1
thr
5*ATP+CO2+3*H2O+NADPH+2*NAD+NH4+Rib5P+gln=>5*ADP +
cyt
9*H+ 2*NADH + NADP + 6*Pi +aKG + his
10*ATP+CO2+9*H2O+2*METHF+3*NAD+Rib5P+
cyt
asp+3*gln+gly=>10*ADP+GMP+17*H+3*NADH+10*Pi+2*THF+fum
+3*glu
9*ATP+CO2+6*H2O+2*METHF+2*NAD+Rib5P+
cyt
2*asp+2*gln+gly=>9*ADP+AMP+15*H+2*NADH+9*Pi+2*THF+2*fu
m+2*glu
6*ATP+2*H2O+NAD+Rib5P+asp+gln=>6*ADP+5*H+NADH+4*Pi+U cyt
TP+glu
ATP+H2O+2*g6P=>ADP+H+2*Pi+t6P
cyt
g6P = g1P
cyt
0.167*ATP + 0.167*g1P + 0.167*H2O => 0.167*ADP + 0.167*H +
cyt
0.333*Pi + psacch
tre_cyt=>tre_external
0.0107700000*f6P_cytosol+0.4379728400*ATP_cytosol+0.365703983*H2O_
cytosol+0.2655890000*psacch_cytosol+0.0917300000*NADPH_cytosol+0.06
49910000*AcCoA_cytosol+0.0152493360*gln_cytosol+0.0115986120*NADH
_cytosol+0.0110120430*ala_cytosol+0.0092606460*gly_cytosol+0.007813000
0*O2_cytosol+0.0070587270*val_cytosol+0.0070498970*leu_cytosol+0.0069
406120*ser_cytosol+0.0056149110*thr_cytosol+0.0053281660*pro_cytosol+0.
0052570280*arg_cytosol+0.0050031770*asp_cytosol+0.0044570850*lys_cyto
sol+0.0043950040*ile_cytosol+0.0042606890*phe_cytosol+0.0042441450*me
t_cytosol+0.0036256830*asn_cytosol+0.0024294300*his_cytosol+0.00226900
00*GAP_cytosol+0.0021845980*tyr_cytosol+0.0018666600*AMP_cytosol+0.
0013906350*GMP_cytosol+0.0012400000*E4P_cytosol+0.0011873880*Rib5
P_cytosol+0.0010060000*THF_cytosol+0.0008985640*CTP_cytosol+0.00089
00680*trp_cytosol+0.0004450030*cys_cytosol+0.0004030000*g6P_cytosol=>
0.0010060000*MYTHF_cytosol+0.0030300000*homcys_cytosol+0.00303140
50*glu_cytosol+0.0046320000*CO2_cytosol+0.0115986120*NAD_cytosol+0.
0649910000*HCoA_cytosol+0.0917300000*NADP_cytosol+0.3411103560*H
_cytosol+0.4379728400*ADP_cytosol+0.452434968*Pi_cytosol+biomass_exte
rnal
8*ATP + 4*H2O + O2 + cys + val + PAA => 8*ADP + 8*H + 8*Pi +
cyt
penG
0.0013
0.0007
0.0009
0.0004
0.0005
0.0347
0.2076
0.0005
0.4928
0.0032
Table B. 3: Metabolic Reaction Network of penGv4 model
r1.2
r1.3
r1.4
r1.9
l1.1
l1.2
l1.3
r2.1
r2.2
r2.3
r2.4
r2.5
r2.6
Reactions
g6P = f6P
ATP + f6P => ADP + f16P + H
f16P = 2*GAP
ADP + H + PEP => ATP + pyr
ATP:cyt+ H:ext+ glc:ext => ADP:cyt + 2*H:cyt + g6P:cyt
GAP+NAD+Pi+ADP = 3PG + ATP + H + NADH
3PG = H2O + PEP
g6P + H2O + NADP => 6Pgluct + 2*H + NADPH
6Pgluct + NADP => CO2 + NADPH + Ribu5P
Ribu5P = Rib5P
Ribu5P = Xylu5P
Rib5P + Xylu5P = GAP + sed7P
GAP + sed7P = E4P + f6P
μmol/gDW/s
0.0607
0.1138
0.1138
0.2314
0.1911
0.2539
0.2397
0.0944
0.0944
0.0360
0.0584
0.0316
0.0316
- 12 -
r2.7
r4.1
r4.2
r4.3
r4.4
r4.7
r4.8
r4.9
r4.10
r4.11
r5.1
l6.7
l6.8
r8.1
r8.2
R9.1
R9.2
R9.4
R9.6
R9.7
R9.8
R9.11
R9.13
R9.14
R9.15
R9.20
r11.1
r11.2
r11.3
r11.7
r11.8
r11.9
r11.10
r11.16
r11.17
r11.20
r11.21
r11.22
r11.34
l11.1
l11.2
l11.3
E4P + Xylu5P = f6P + GAP
HCoA + NAD + pyr => AcCoA + CO2 + NADH
AcCoA + H2O + OAA = citr + H + HCoA
citr = iCitr
iCitr + NAD => aKG + CO2 + NADH
aKG + HCoA + NAD => CO2 + NADH + succCoA
ADP + Pi + succCoA = ATP + HCoA + succ
FAD + succ = FADH2 + fum
fum + H2O = mal
mal + NAD = H + NADH + OAA
ATP + CO2 + H2O + pyr => ADP + 2*H + OAA + Pi
1.104*ADP+1.104*H+FADH2+0.5*O2+1.104*Pi=>1.104*ATP+2.104*H2O+
FAD
1.87289439*ADP+2.87289439*H+NADH+0.5*O2+1.87289439*Pi=>1.87289
439*ATP+2.87289439*H2O+NAD
CO2 + METHF + NADH + NH4= gly + NAD + THF
METHF + NADH + H => MYTHF + NAD
ATP:cyt + H2O:cyt = ADP:cyt + H:ext + Pi:cyt
2*H:ext + Pi:ext => 2*H:cyt + Pi:cyt
2*H:ext + SO4:ext = 2*H:cyt + SO4:cyt
O2:ext = O2:cyt
CO2:cyt = CO2:ext
penG:cyt => penG:ext
PAA:ext => PAA:cyt
ExPept:cyt => ExPept:ext
psacch:cyt => psacch:ext
H2O:cyt = H2O:ext
H:ext + NH4:ext = H:cyt + NH4:cyt
aKG + H + NADPH + NH4 = glu + H2O + NADP
ATP + glu + NH4 = ADP + gln + Pi
ATP + glu + H + 2*NADPH => ADP + H2O + 2*NADP + Pi + pro
AcCoA + glu + H2O + NAD => aAd + CO2 + HCoA + NADH
aAd + 2*ATP + glu + H2O + NAD + 2*NADPH => 2*ADP + aKG + H + lys +
NADH + 2*NADP + 2*Pi
3PG + glu + H2O + NAD => aKG + H + NADH + Pi + ser
ser + THF => gly + H2O + METHF
glu + OAA = aKG + asp
asp + 2*ATP + H2O + NH4 => 2*ADP + asn + H + 2*Pi
homcys + MYTHF = met + THF
glu + 2*H + NADPH + pyr + thr => aKG + CO2 + H2O + ile + NADP + NH4
glu + pyr = aKG + ala
0.134*ala + 0.07*arg + 0.0386*asn + 0.0386*asp + 0.02*cys + 0.0509*gln +
0.0509*glu + 0.132*gly + 0.0119*his + 0.0307*ile + 0.0559*leu + 0.0348*lys
+ 0.0103*met + 0.0266*phe + 0.0537*pro + 0.044*ser + 0.0467*thr + 0.01*trp
+ 0.0203*tyr + 0.12*val => 4.53*ExPept + H2O
2*H + NADPH + glu + 2*pyr => CO2 + H2O + NADP + aKG + val
AcCoA + H + NADPH + NAD + glu + 2*pyr => 2*CO2 + HCoA + NADH +
NADP + aKG + leu
3*ATP+E4P+NADPH+2*PEP+Rib5P+gln+ser=>3*ADP + CO2 + GAP + H2O
+ 3*H + NADP + 6*Pi + glu + pyr +trp
0.0268
0.1714
0.1329
0.1329
0.1329
0.1170
0.1170
0.1170
0.1228
0.1228
0.0277
0.1170
0.8208
0.0013
0.0017
0.3868
0.0038
0.0043
0.4760
0.5080
0.0032
0.0032
0.0426
0.0767
0.7457
0.0994
0.0793
0.0179
0.0031
0.0025
0.0025
0.0142
0.0062
0.0175
0.0021
0.0022
0.0025
0.0067
0.0094
0.0078
0.0040
0.0005
- 13 -
l11.4
l11.5
l11.7
l11.8
l11.9
l11.10
l11.11
l11.12
r13.6
l13.2
l13.3
l13.4
r15.1
r17.7
l17.1
l17.3
l17.4
l17.6
l18.2
l19.1
ATP+E4P+NADPH+NAD+2*PEP+glu=>ADP+CO2+NADH+NADP+4*Pi+a
KG+tyr
ATP+E4P+H+NADPH+2*PEP+glu=>ADP +CO2 + H2O + NADP +
4*Pi+aKG+phe
4*ATP + 2*H + 4*NADPH + SO4 + ser => 4*ADP + H2O + 4*NADP + 4*Pi
+ cys
5*ATP + 4*H + 6*NADPH + SO4 + asp => 5*ADP+ H2O + 6*NADP + 5*Pi
+ homcys
2*ATP+ H2O + H + 2*NADPH + asp => 2*ADP + 2*NADP + 2*Pi + thr
5*ATP+CO2+3*H2O+NADPH+2*NAD+NH4+Rib5P+gln=>5*ADP + 10*H+
2*NADH + NADP + 6*Pi +aKG + his
ATP+2*glu+NADPH+H=>ADP+aKG+orn+NADP+Pi
4*ATP+CO2+gln+3*H2O+orn+asp=> 4*ADP+glu+7*H+4*Pi+arg+fum
ATP + gln + H2O + UTP => ADP + CTP + glu + 2*H + Pi
10*ATP+CO2+9*H2O+2*METHF+3*NAD+Rib5P+
asp+3*gln+gly=>10*ADP+GMP+19*H+3*NADH+10*Pi+2*THF+fum+3*glu
9*ATP+CO2+6*H2O+2*METHF+2*NAD+Rib5P+
2*asp+2*gln+gly=>9*ADP+AMP+16*H+2*NADH+9*Pi+2*THF+2*fum+2*g
lu
6*ATP+2*H2O+NAD+Rib5P+asp+gln=>6*ADP+6*H+NADH+4*Pi+UTP+gl
u
ATP + H2O => ADP + H + Pi
H2O + t6P => Pi + tre
ATP+H2O+2*g6P=>ADP+H+2*Pi+t6P
g6P = g1P
0.167*ATP + 0.167*g1P + 0.167*H2O => 0.167*ADP + 0.167*H + 0.333*Pi +
psacch
tre:cyt=>tre:ext
0.0107700000*f6P_cytosol+0.4379728400*ATP_cytosol+0.365703983*H2O_
cytosol+0.2655890000*psacch_cytosol+0.0917300000*NADPH_cytosol+0.06
49910000*AcCoA_cytosol+0.0152493360*gln_cytosol+0.0115986120*NADH
_cytosol+0.0110120430*ala_cytosol+0.0092606460*gly_cytosol+0.007813000
0*O2_cytosol+0.0070587270*val_cytosol+0.0070498970*leu_cytosol+0.0069
406120*ser_cytosol+0.0056149110*thr_cytosol+0.0053281660*pro_cytosol+0.
0052570280*arg_cytosol+0.0050031770*asp_cytosol+0.0044570850*lys_cyto
sol+0.0043950040*ile_cytosol+0.0042606890*phe_cytosol+0.0042441450*me
t_cytosol+0.0036256830*asn_cytosol+0.0024294300*his_cytosol+0.00226900
00*GAP_cytosol+0.0021845980*tyr_cytosol+0.0018666600*AMP_cytosol+0.
0013906350*GMP_cytosol+0.0012400000*E4P_cytosol+0.0011873880*Rib5
P_cytosol+0.0010060000*THF_cytosol+0.0008985640*CTP_cytosol+0.00089
00680*trp_cytosol+0.0004450030*cys_cytosol+0.0004030000*g6P_cytosol=>
0.0010060000*MYTHF_cytosol+0.0030300000*homcys_cytosol+0.00303140
50*glu_cytosol+0.0046320000*CO2_cytosol+0.0115986120*NAD_cytosol+0.
0649910000*HCoA_cytosol+0.0917300000*NADP_cytosol+0.3411103560*H
_cytosol+0.4379728400*ADP_cytosol+0.452434968*Pi_cytosol+biomass_fer
mentor
8*ATP + 4*H2O + O2 + cys + val + PAA => 8*ADP + 8*H + 8*Pi + penG
0.0013
0.0023
0.0036
0.0007
0.0057
0.0013
0.0032
0.0032
0.0004
0.0007
0.0009
0.0004
1.1664
0.0005
0.0005
0.0347
0.2076
0.0005
0.4928
0.0032
- 14 -
Appendix C: Connectivity metabolic network
The information presented in this appendix is organized in the following matter:
Code:
reaction number, e.g. r12.1 or in case of partial reactions r12.1b. The reaction numbers are denoted
as in (van Gulik et al. 2000). In case of a lumped reaction the reaction number is denoted with a l,
for example l1.2.
Enzyme:
recommend name of the enzyme
EC number:
EC number
Reaction Brenda: (partial) reaction as in the Brenda database
Reversibility:
whether the reaction is reversible. This is based on the KEGG database unless specified.
Act & Inh:
activators & inhibitors, which are presented in tables
Eff. NAS:
activators & inhibitors which are not included in the stoichiometric model
Turnover nr:
turnover rate of metabolites in s-1
Spec activity:
specific activity in μmol/min/mg
Mechanism:
mechanistic rate equations
Location:
location of the reaction according to (van Gulik et al. 2000)
8
Lumped metabolites
The metabolites AMP and diphosphosphate, which is also known as pyrophosphate, are involved in many catalyzed
reactions but have (largely) been eliminated from the stoichiometric model derived by(van Gulik et al. 2000). The
reactions that are discussed in this paragraph have been used to lump these compounds to the metabolites that have
been included in the stoichiometric model.
Code:
l1
Enzyme:
adenylate kinase
EC number:
2.7.4.3
Reaction Brenda: AMP + ATP => 2 ADP
Reversibility:
reversible
Act & Inh:
none found in Brenda database
Eff. NAS:
5,5'-dithiobis(2-nitrobenzoic acid) (Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
1900 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
Code:
l2
Enzyme:
inorganic diphosphatase
EC number:
3.6.1.1
Reaction Brenda: PP + H2O => 2 Pi
Reversibility:
irreversible
Act & Inh:
Table 8.1
Eff. NAS:
none found
Turnover nr:
260 s-1 (Saccharomyces cerevisiae)
Spec activity:
13.1 μmol/min/mg (Aspergillus oryzae)
Mechanism:
unknown
Location:
soluble and membrane bound in mitochondria (Saccharomyces cerevisiae)
Table 8.1: Connectivity for inorganic diphosphatase
effector
organism
comment
- 15 -
ATP
glu
g6P
PP
Saccharomyces cerevisiae
Aspergillus oryzae
Aspergillus oryzae
Aspergillus oryzae
competitive inhibitor; turnover number <0.005 s-1
competitive inhibitor
competitive inhibitor
Km = 0.55 mM (pH = 3)
In the stoichiometric model 3’-phosphoadenosine-5’-phosphosulphate (PAPS) is employed as an activated carbon
source. The result is that enzymes that are involved in the sulfur assimilation pathway produce the side product PAP.
An intracellular accumulation may lead to the inhibition of these enzymes in yeast. Furthermore since it imitates the
monomers of polyribonucleotide chains, it also negatively influences the RNA-processing [5].
Code:
l3
Enzyme:
3'(2'),5'-bisphosphate nucleotidase
EC number:
3.1.3.7
Reaction Brenda: H2O + PAP => AMP + Pi
Reversibility:
unknown
Act & Inh:
none found
Eff. NAS:
inositol 1,4-bisphophate (Schizosaccharomyces pombe)
Turnover nr:
unknown
Mechanism:
unknown
Location:
unknown
9
Glycolysis
r1.1: glc + ATP => g6P + ADP + H
Glucose can be phosphorylated by three enzymes: glucokinase and hexokinase PI & PII. In the models of Galazzo et
al [6] and Hynne et al [7] only the substrates glc and ATP are considered to influence the activity whereas Teusink et
al [8] extends his model to include the influence of the products. In all these models the influence of citrate, 3PG and
phosphate is neglected.
Code:
r1.1a
Enzyme:
hexokinase & glucokinase
EC number:
2.7.1.1 (hexokinase) & 2.7.1.2 (glucokinase)
Reaction Brenda: glc + ATP => g6P + ADP + H
Reversibility:
irreversible
Act & Inh:
Table 9.1
Eff. NAS:
xylose, mannose and fructose [1], 6-aminoglucose (1.65<Ki<4.7 mM) [4] (Saccharomyces
cerevisiae)
Turnover nr:
unknown
Spec activity:
40 μmol/min/mg on glucose (Aspergillus niger)
Mechanism:
(1) competitive inhibition v = Vmax*Xglc/(Km*(1+XAMG/KAMG)+Xglc) [4]
(2) reversible Michaelis-Menten kinetics for two non-competiting substrate-product couples [8]
Location:
cytosol
Table 9.1: Connectivity for hexokinase
effector
organism
ATP
Saccharomyces cerevisiae
Aspergillus niger
ADP
Aspergillus niger
3PG
Saccharomyces cerevisiae
citr
Saccharomyces cerevisiae
Aspergillus niger
Pi
Saccharomyces cerevisiae
comment
activates but strong inhibition at high phys. conc.
Km = 0.54 mM
inhibitor
activates
allosteric activator
noncompetitive inhibitor to glucose and ATP; Ki = 0.15
mM
activates
- 16 -
glc
Saccharomyces cerevisiae
Aspergillus niger
0.04<Km<0.12 mM [1]; 0.25<Km<0.31 mM [4]
Km = 0.023 mM
r1.2: g6P => f6P
This reaction can be described with a reversible Michaelis Menten kinetic for a single substrate and product [7, 8]. In
these models the influence of effectors presented in Table 9.2 is neglected.
Code:
r1.2
Enzyme:
glucose-6-phosphate isomerase
EC number:
5.3.1.9
Reaction Brenda: g6P => f6P
Reversibility:
reversible
Act & Inh:
Table 9.2
Eff. NAS:
mannose-6P and man-6P [1], 2-chloro-2-deoxy-D-glucose 6-phosphate (Ki = 0.27 mM), 2-deoxy2-fluoro-D-glucose 6-phosphate (Ki = 11.3 mM), 2-deoxy-D-arabino-hexose 6-phosphate (Ki = 1.9
mM), D-glucosamine 6-phosphate (Ki = 2.9 mM), [4] (Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
(1) v = (Vmax*Xg6P)/(Km+Xg6P) [4]
(2) v = (Vmax* Xg6P)/(Km*(1+I/Ki)+ Xg6P) [4]
(3) v = kcat*E* Xg6P (Km+ Xg6P); v = kcat*E* Xg6P /(Km+ Xg6P) [4]
Location:
cytosol
Table 9.2: Connectivity for glucose-6-phosphate isomerase
effector
organism
comment
6Pgluct
Saccharomyces cerevisiae
inhibitor
g6P
Saccharomyces cerevisiae
0.3<Km<1.5 mM [1], 0.81<Km<1.03 mM [4]
f6P
Saccharomyces cerevisiae
0.11<Km<0.23
E4P
Saccharomyces cerevisiae
inhibitor
Xylu5P
Saccharomyces cerevisiae
inhibitor
Rib5P
Saccharomyces cerevisiae
inhibitor
Ribu5P
Saccharomyces cerevisiae
inhibitor
Sed7P
Saccharomyces cerevisiae
inhibitor
r1.3: f6P + ATP => f16P + ADP + H
Code:
r1.3
Enzyme:
6-phosphofructokinase
EC number:
2.7.1.11
Reaction Brenda: f6P + ATP => f16P + ADP + H
Reversibility:
irreversible
Act & Inh:
Table 9.3
Eff. NAS:
f26P (activator in Saccharomyces cerevisiae & Kluyveromyces lactis) and f1P (inhibitor in
Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
between 60 & 180 μmol/min/mg (Saccharomyces cerevisiae), 136 μmol/min/mg
(Schizosaccharomyces pombe)
Mechanism:
unknown
Location:
cytosol
Table 9.3: Connectivity for 6-phosphofructokinase
effector
organism
comment
ADP
Aspergillus niger
activates at high f6P conc., inhibits at low f6P conc.
Saccharomyces cerevisiae
slight activation
- 17 -
AMP
ATP
citr
g6P
NH4
Pi
PEP
Aspergillus niger
Saccharomyces cerevisiae
Kluyveromyces lactis
Aspergillus niger
Saccharomyces cerevisiae
Kluyveromyces lactis
Aspergillus niger
Saccharomyces cerevisiae
Aspergillus niger
Aspergillus niger
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Aspergillus niger
activates at low f6P conc., inhibits at high f6P conc.
activation
activation
inhibitor, synergistic with citrate
inhibitor
inhibitor
inhibits strongly, synergistic with ATP, Pi & AMP, NH4
inhibits, activity can be restored by cAMP, ADP or f16P
activates
activates, counteracts the inhibition by ATP,
enhances the affinity for f6P
activator, synergistic with AMP
Ki = 0.15 mM
r1.4: f16P => 2 GAP
Fructose-bisphosphate aldolase catalyzes the equilibrium reaction of f16P into GAP and DHAP. Since both DHAP
and GAP are in equilibrium, the triosephosphate isomerase reaction will be modeled as an equilibrium block,
analogously to Teusink’s kinetic model [8] and the stoichiometric model developed by van Gulik et al [9].
The Brenda database does not contain any effectors for this enzyme. The reaction can be described by an ordered
uni-bi mechanism that is a function of GAP, DHAP and f16P [8].
Code:
r1.4
Enzyme:
fructose-bisphosphate aldolase
EC number:
4.1.2.13
Reaction Brenda: f16P => 2 GAP
Reversibility:
reversible
Act & Inh:
none found in Brenda database
Eff. NAS:
EDTA (Saccharomyces sp.)
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
r1.5: GAP + NAD + Pi => 13PG + NADH + H
Glyceraldehyde-3-phosphate dehydrogenase is the most abundant enzyme in yeast. According to Teusink the
reaction can be described by a reversible Michaelis-Menten kinetic equation for two non-competing substrateproduct couples.
Code:
r1.5
Enzyme:
glyceraldehyde-3-phosphate dehydrogenase
EC number:
1.2.1.12
Reaction Brenda: GAP + NAD + Pi => 13PG + NADH + H
Reversibility:
reversible
Act & Inh:
Table 9.4
Eff. NAS:
none found in Brenda database
Turnover nr:
16.7 s-1 (Saccharomyces cerevisiae)
Spec activity:
unknown
Mechanism:
v = (Vmax*XNAD/(1+KmNAD))+(XGAP*Vmax/KmGAP) mM [4]
Location:
cytosol
Table 9.4: Connectivity for glyceraldehyde-3-phosphate dehydrogenase
effector
organism
comment
GAP
Saccharomyces cerevisiae
Km = 0.6 mM [1]; Km = 0.21 mM [4]
NAD
Saccharomyces cerevisiae
Km = 0.1 mM [1]; Km = 0.09 mM [4]
- 18 -
Pi
13PG
NADH
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Km = 1.5 mM
Km = 1 mM [4]
Km = 0.059 mM [4]
r1.6: 13PG + ADP => 3PG + ATP
Code:
r1.6
Enzyme:
phosphoglycerate kinase
EC number:
2.7.2.3
Reaction Brenda: 13PG + ADP => 3PG + ATP
Reversibility:
reversible
Act & Inh:
Table 9.5
Eff. NAS:
1,4-bis(difluoro)-1,4-diphospho-diethylether, 1,5-bisphosphonopentane, 1,4-bisphosphonobutane,
2-hydroxy-3,5-diiodobenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), inositol triphosphate, pchloromercuribenzoate (Saccharomyces cerevisiae)
Turnover nr:
354 s-1 (Saccharomyces cerevisiae), 3.4 s-1 (Saccharomyces cerevisiae mutant P204H)
Spec activity:
745-945 mmol/min/mg purified enzyme of Saccharomyces cerevisia, 468 mmol/min/mg purified
enzyme of wild type Saccharomyces cerevisia whereas for the mutant P204H the specific activity
equals 4.5 μmol/min/mg. The P204F mutants has a specific activity of 1.4 μmol/min/mg.
Mechanism:
(1) v = (Vmax*X3PG)/(Km+X3PG) [4]
(2) v = (Vmax *A*B)/(Kia*Kb+Ka*B+Kb*A+A*B); A = ATP, B = 3PG, [4]
(3) v = (Vmax *A)/(K*(1+I/Kis)+A); A = 0.02 mM; B = ATP, I = CrATP [4].
Location:
cytosol
Table 9.5: Connectivity for phosphoglycerate kinase
effector
organism
comment
AS
Saccharomyces cerevisiae
inhibition at high conc., activation at low conc.
13PG
Saccharomyces cerevisiae
product inhibitor reverse reaction
3PG
Saccharomyces cerevisiae
0.7<Km<0.28 mM [1]; 0.08< Km< 6.56 mM [4], Ki =
0.37 mM
ATP
Saccharomyces cerevisiae
Km = 0.33 mM [1]; 0.14<Km< 0.51 mM [4]; Ki = 0.7
mM
ADP
Saccharomyces cerevisiae
inhibitor, competitive to 3PG
AMP
Saccharomyces cerevisiae
inhibitor
Pi
Saccharomyces cerevisiae
inhibitor, above 0.5mM competitive for 3PG
SO42Saccharomyces cerevisiae
inhibits at 3PG conc. below 0.5-1 mM, activates at
higher substrate concentrations
Scitr
Saccharomyces cerevisiae
inhibits at high conc., acceleration of activity at low
conc.
Ssucc
Saccharomyces cerevisiae
inhibits at high conc., acceleration of activity at low
conc.
r1.7: 3PG => 2PG
Although the activity of PGM depends on 2,3-phosphoglycerate, it is assumed the enzyme is saturated with this
compound.
Code:
r1.6
Enzyme:
phosphoglycerate mutase
EC number:
5.4.2.1
Reaction Brenda: 3PG => 2PG
Reversibility:
reversible
Act & Inh:
Table 9.6
- 19 -
Eff. NAS:
Turnover nr:
Spec activity:
Mechanism:
Location:
2,3-butanedione, 8-hydroxyquinoline 5-sulfonic acid, benzene-carboxylate derivates,
diethyldicarbonate, EDTA , 2,3PG, 2-phosphoglycollate (Saccharomyces cerevisiae), EDTA
(Aspergillus nidulans)
384-530 s-1 (Saccharomyces cerevisiae). Turnover numbers of mutants of Saccharomyces
cerevisiae are 324 s-1 (K246G), 20 s-1 (S11A), 4.7 s-1 (E86Q), 2.1 s-1 (S11G) & 0.078 s-1 (H181A).
Turnover number for Schizosaccharomyces pombe is 82 s-1.
1280 μmol/min/mg (Saccharomyces cerevisiae) and 210-218 μmol/min/mg (Schizosaccharomyces
pombe),
ping-pong mechanism via a phosphorylated His-intermediate [1]
cytosol
Table 9.6: Connectivity for phosphoglycerate mutase
effector
organism
comment
inoh
Saccharomyces cerevisiae
inhibitor, Ki = 0.004 mM
2PG
Saccharomyces cerevisiae
Km = 0.041 mM
3PG
Saccharomyces cerevisiae
0.23<Km<0.74 mM
r1.8: 3PG => PEP + H2O
The reaction can be described with the same kinetics as with phosphoglycerate mutase.
Code:
r1.8
Enzyme:
phosphopyruvate hydratase
EC number:
4.2.1.11
Reaction Brenda: 3PG => PEP + H2O
Reversibility:
reversible
Act & Inh:
Table 9.7
Eff. NAS:
D-erythro-2,3-dihydroxybutyric
acid
3-phosphate,
D-glyceric
acid
3-phosphate,
phosphonoacetohydroxamate (Saccharomyces cerevisiae)
Turnover nr:
78-230 s-1 (wild type Saccharomyces cerevisiae), 0.018-0.019 s-1 (S39A mutant of Saccharomyces
cerevisiae)
Spec activity:
442 μmol/min/mg (wild type Saccharomyces cerevisiae). Specific activities of the mutants of
Saccharomyces cerevisiae are 248 μmol/min/mg (N207A), 3.3 μmol/min/mg (H159A), 1.9
μmol/min/mg (H159F) and 1.1 μmol/min/mg (H159N). The specific activity of several members
of the genus Candida is between 60 and 70 μmol/min/mg.
Mechanism:
unknown
Location:
cytosol
Table 9.7: Connectivity for phosphopyruvate hydratase
effector
organism
comment
Pi
Saccharomyces cerevisiae
inhibitor
2PG
Saccharomyces cerevisiae
0.031<Km<0.3 mM
Phosphopyruvate hydratase can also catalyse the partial reaction denoted as r11.27c [4].
r1.9: PEP + ADP + H => pyr + ATP
Although pyruvate kinase is strongly activated by f16P, the maximum activation is achieved at the f16P
concentration of 5mM. If the f16P concentration is significant higher the model can be simplified to a MichaelisMenten model such as for hexokinase.
Code:
r1.9
Enzyme:
pyruvate kinase
EC number:
2.7.1.40
Reaction Brenda: PEP + ADP + H => pyr + ATP
- 20 -
Reversibility:
Act & Inh:
Eff. NAS:
Turnover nr:
Spec activity:
Mechanism:
Location:
irreversible
Table 9.8
IDP (Saccharomyces cerevisiae)
unknown
250-340 μmol/min/mg (Saccharomyces cerevisiae)
sigmoidal kinetics with respect to PEP (Schizosaccharomyces pombe)
cytosol
Table 9.8: Connectivity for phosphopyruvate hydratase
effector
organism
comment
ADP
Saccharomyces cerevisiae
Km = 0.16 mM [1]
PEP
Saccharomyces cerevisiae
Km = 0.099 mM [1]; 0.3<Km<10 mM [4]
f16P
Saccharomyces cerevisiae
activator
10 Pentose phosphate pathway
r2.1: g6P + H2O + NADP => 6Pgluct + 2 H + NADPH
Glucose-6-phosphate isomerase has already been elaborated in chapter 9 for reaction r1.2. However in the pentose
phosphate pathway it catalyzes a different reaction, see r2.1a. In reaction r2.1b, β-glucose-6-phosphate is converted
into D-glucono-1,5-lactone 6-phosphate which subsequently is used to produce 6Pgluc, see r2.1c.
Code:
r2.1a
Enzyme:
glucose-6-phosphate isomerase
EC number:
5.3.1.9
Reaction Brenda: α-g6P => β-g6P
Reversibility:
reversible
Act & Inh:
see r1.2
Eff. NAS:
see r1.2
Turnover nr:
see r1.2
Spec activity:
see r1.2
Mechanism:
unknown
Location:
cytosol
Code:
r2.1b
Enzyme:
glucose-6-phosphate 1-dehydrogenase
EC number:
1.1.1.49
Reaction Brenda: β-g6P + NADP => D-glucono-1,5-lactone 6-phosphate + NADPH + H
Reversibility:
irreversible
Act & Inh:
Table 10.1
Eff. NAS:
none found in the Brenda database
Turnover nr:
unknown
Spec activity:
745 mmol/min/mg (Aspergillus nidulans)
Mechanism:
unknown
Location:
cytosol
Table 10.1: Connectivity for glucose-6-phosphate 1-dehydrogenase
effector
organism
comment
g6P
Aspergillus niger
Km = 0.153 mM
NADP
Aspergillus niger
Km = 0.026 mM
NADPH
Aspergillus niger
Km = 0.02 mM
Code:
Enzyme:
r2.1c
6-phosphogluconolactonase
- 21 -
EC number:
3.1.1.31
Reaction Brenda: 6-phospho-D-glucono-1,5-lactone + H2O => 6Pgluct
Reversibility:
irreversible
Act & Inh:
none found in the Brenda database
Eff. NAS:
none found in the Brenda database
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
r2.2: 6Pgluct + NADP => CO2 + NADPH + Ribu5P
Code:
r2.2
Enzyme:
phosphogluconate dehydrogenase (decarboxylating)
EC number:
1.1.1.44
Reaction Brenda: 6Pgluct + NADP => CO2 + NADPH + Ribu5P
Reversibility:
reversible (Candida utilis & Neurospora crassa) [1], irreversible [2]
Act & Inh:
Table 10.2
Eff. NAS:
many effectors given such as 5-(dimethylamine)naphthalin-1-sulfonylchloride, 5,5'-dithiobis(2nitrobenzoic acid) , butan-2,3-dione, arsenate and pyridoxal 5'-phosphate (Candida utilis)
Turnover nr:
unknown
Spec activity:
12-48 μmol/min/mg (Candida utilis), 30-30.8 μmol/min/mg (Neurospora crassa), 39.4
μmol/min/mg (Schizosaccharomyces p.)
Mechanism:
unknown
Location:
cytosol
Table 10.2: Connectivity for phosphogluconate dehydrogenase (decarboxylating)
effector
organism
comment
6Pgluc
Saccharomyces cerevisiae
0.068<Km<3.13 mM
Candida biodinii
Km = 0.011 mM
Candida utilis
0.0068<Km<0.64 mM, pH dependent
inhibition
NADPH
Candida biodinii & utilis
inhibition
Neurospora crassa
competitive inhibition
Schizosaccharomyces p.
0.025<Km<0.033 mM
NADP
Saccharomyces cerevisiae
0.008<Km<0.02 mM
Candida utilis
Km=0.013 mM
Candida biodinii
ATP
Candida boidinii
inhibitor
f16P
Candida boidinii
inhibitor
Neurospora crassa
inhibitor
Pi
Neurospora crassa
inhibitor
Ribu5P
Candida biodinii & utilis
inhibitor
Schizosaccharomyces p.
inhibitor
SO4
Candida utilis
inhibitor
UTP
Candida biodinii
inhibitor
f6P
Candida utilis
slight activation
CO2
Candida utilis
Km = 50 mM
NAD
Candida utilis
1<Km<114 mM, pH dependent
r2.3: Ribu5P => Rib5P
Code:
Enzyme:
r2.3
ribose-5-phosphate isomerase
- 22 -
EC number:
5.3.1.6
Reaction Brenda: Ribu5P => Rib5P
Reversibility:
reversible
Act & Inh:
Table 10.3
Eff. NAS:
GMP, EDTA, iodoacetamide, NEM and PCMB (Candida utilis)
Turnover nr:
unknown
Spec activity:
356 μmol/min/mg (Candida utilis)
Mechanism:
unknown
Location:
cytosol
Table 10.3: Connectivity for ribose-5-phosphate isomerase
effector
organism
comment
Rib5P
Saccharomyces cerevisiae
Km=0.74 mM
Candida utilis
0.108<Km<2.5 mM
AMP
Candida utilis
competitive inhibitor
UMP
Candida utilis
inhibitor
6Pgluct
Candida utilis
competite inhibitor
r2.4: Ribu5P => Xylu5P
Code:
r2.4
Enzyme:
ribulose-phosphate 3-epimerasee
EC number:
5.1.3.1
Reaction Brenda: Ribu5P => Xylu5P
Reversibility:
reversible
Act & Inh:
Table 10.4
Eff. NAS:
none found
Turnover nr:
1300 s-1 for both the forward and reverse reaction (Saccharomyces cerevisiae).
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Table 10.4: Connectivity for ribulose-phosphate 3-epimerasee
effector
organism
comment
Ribu5P
Saccharomyces cerevisiae
1.5<Km<15 mM
r2.5: Rib5P + Xylu5P => GAP + sed7P
Code:
r2.5
Enzyme:
transketolase
EC number:
2.2.1.1
Reaction Brenda: Rib5P + Xylu5P => GAP + sed7P
Reversibility:
reversible
Act & Inh:
Table 10.5
Eff. NAS:
EDTA, N-acetylimidazole, hydroxypyruvate, L-erythrulose, thiamine diphosphate and
oxythiamine diphosphate (Saccharomyces cerevisiae).
Turnover nr:
the turnover number in Saccharomyces cerevisiae for substrates such as E4P, Rib5P, Xylu5P are
69 s-1, 46-56.7 s-1, 56.7 s-1 respectively.
Spec activity:
18-24 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
Table 10.5: Connectivity for transketolase
- 23 -
effector
Pi
SO4
Rib5P
Xylu5P
GAP
f6P
organism
Saccharomyces cerevisiae
Candida utilis
Saccharomyces cerevisiae
Candida utilis
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
comment
inhibitor
inhibitor
inhibitor
inhibitor
0.093<Km<7 mM
0.023<Km<4.08 mM
Km = 4.9 mM
Km = 1.8 mM
r2.6: GAP + sed7P => E4P + f6P
Code:
r2.6
Enzyme:
transaldolase
EC number:
2.2.1.2
Reaction Brenda: GAP + sed7P => E4P + f6P
Reversibility:
reversible
Act & Inh:
Table 10.6
Eff. NAS:
tetranitromethane & PP (Candida utilis)
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Table 10.6: Connectivity for transketolase
effector
organism
Pi
Saccharomyces cerevisiae
Candida utilis
SO4
Saccharomyces cerevisiae
Candida utilis
f6P
Candida utilis
Saccharomyces cerevisiae
E4P
Candida utilis
Saccharomyces cerevisiae
GAP
Saccharomyces cerevisiae
sed7P
Saccharomyces cerevisiae
comment
together with PP inhibits
together with PP inhibits
inhibitor
inhibitor
Km = 0.8 mM
Km = 0.32 mM
Km = 0.02 mM
Km = 0.018 mM
Km = 0.22 mM
Km = 0.18 mM
r2.7: E4P + Xylu5P => f6P + GAP
Code:
r2.5
Enzyme:
transketolase
EC number:
2.2.1.1
Reaction Brenda: E4P + Xylu5P => f6P + GAP
Reversibility:
reversible
Act & Inh:
Table 10.5
Eff. NAS:
see r2.5
Turnover nr:
unknown
Spec activity:
see r2.5
Mechanism:
see r2.5
Location:
cytosol
11 TCA cycle
r4.1: HCoA + NAD + pyr => AcCoA + CO2 + NADH
- 24 -
Code:
r4.1a
Enzyme:
pyruvate dehydrogenase (acetyl-transferring)
EC number:
1.2.4.1
Reaction Brenda: pyr + ThPP + lipoamide => S-acetyldihydrolipoamide + CO2
Reversibility:
irreversible
Act & Inh:
Table 11.1
Eff. NAS:
2-p-toluidinonaphthalene-6-sulfonate (Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
0.12-0.15 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
mitochondria
Table 11.1: Connectivity for pyruvate dehydrogenase
effector
organism
comment
pyr
Saccharomyces cerevisiae
Km = 0.65 mM
citr
Neurospora crassa
29% inhibition at 0.25 mM, 87% inhibition at 50.0 mM.
ATP
Neurospora crassa
93% inhibition at 5 mM, due to phosphorylation
Saccharomyces cerevisiae
no inhibition
Code:
r4.1b
Enzyme:
dihydrolipoamide S-acetyltransferase
EC number:
2.3.1.12
Reaction Brenda: CoA + S-acetyldihydrolipoamide => dihydrolipoamide + AcCoA
Reversibility:
reversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
mitochondria
Code:
r4.1c
Enzyme:
dihydrolipoamide dehydrogenase
EC number:
1.8.1.4
Reaction Brenda: dihydrolipoamide + NAD => lipoamide + NADH + H
Reversibility:
reversible
Act & Inh:
none found
Eff. NAS:
dihydrolipoamide (Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
843 μmol/min/mg (Saccharomyces cerevisiae), 177 μmol/min/mg (Neurospora crassa)
Mechanism:
unknown
Location:
mitochondria
Table 11.2: Connectivity for dihydrolipoamide dehydrogenase
effector
organism
comment
NADH
Candida krusei
inhibitor
Saccharomyces cerevisiae
inhibitor
NAD
Saccharomyces cerevisiae
Km = 0.15 mM
The overall reaction is:
HCoA + NAD + pyr => AcCoA + CO2 + NADH
r4.2: AcCoA + H2O + OAA => citr + H + HCoA
- 25 -
Code:
r4.2
Enzyme:
citrate (Si)-synthase
EC number:
2.3.3.1
Reaction Brenda: AcCoA + H2O + OAA => citr + HCoA
Reversibility:
reversible (Aspergillus niger), irreversible (Saccharomyces cerevisiae) [1]
Act & Inh:
Table 11.3
Eff. NAS:
propionyl-CoA (Aspergillus nidulans)
Turnover nr:
unknown
Spec activity:
41.5 μmol/min/mg (Aspergillus nidulans); 79 μmol/min/mg purified enzyme (Aspergillus niger) &
50-60 μmol/min/mg (Penicillium spiculisporum)
Mechanism:
unknown
Location:
mitochondria
Table 11.3: Connectivity for citrate (Si)-synthase
effector
organism
comment
ADP
Saccharomyces cerevisiae
inhibitor
strong inhibitor
ATP
Saccharomyces cerevisiae
inhibits, noncompetitive against acetyl-CoA
Yarrowia lipolytica
inhibits, noncompetitive against AcCoA; Ki = 3.5mM
Penicillium spiculisporum
NADH
Saccharomyces cerevisiae
12% inhibition at 5 mM
NADPH
Saccharomyces cerevisiae
40% inhibition at 5 mM
citr
Yarrowia lipolytica
inhibitor
AcCoA
Saccharomyces cerevisiae
Km = 0.004 mM
Yarrowia lipolytica
0.01<Km<10
Penicillium spiculisporum
0.03< Km <0.08 mM
Aspergillus niger
Km=0.014 mM
Km = 0.003 mM
OAA
Saccharomyces cerevisiae
Yarrowia lipolytica
0.005<Km<5 mM
Penicillium spiculisporum
Km=0.07 mM
Aspergillus niger
Km= 0.007 mM
r4.3: citr =>iCitr
This reversible reaction may proceeds via cis-aconitate.
Code:
r4.3
Enzyme:
aconitate hydratase
EC number:
4.2.1.3
Reaction Brenda: (1) citrate => cis-aconitate + H2O;
(2) cis-aconitate + H2O => iCitr
Reversibility:
reversible
Act & Inh:
Table 11.4
Eff. NAS:
oxalomalate, cis-aconitate (Saccharomyces cerevisiae), PCMB, urea (Candida lipolytica),
1,10-phenanthroline, adipate, quinaldic acid, maleate, methyl-cis-aconitate and fluoracetate
(Saccharomyces lipolytica)
Turnover nr:
unknown
Spec activity:
100 μmol/min/mg (Candida lipolytica)
Mechanism:
unknown
Location:
mitochondria
Table 11.4: Connectivity for citrate (Si)-synthase
effector
organism
comment
iCitr
Saccharomyces lipolytica
Km = 0.045 mM
- 26 -
r4.4: iCitr + NAD => aKG + CO2 + NADH
Code:
r4.4
Enzyme:
isocitrate dehydrogenase (NAD+)
EC number:
1.1.1.41
Reaction Brenda: iCitr + NAD => aKG + CO2 + NADH + H
Reversibility:
reversible
Act & Inh:
Table 11.5
Eff. NAS:
beta-mercapto-alpha-ketoglutarate, D-tartrate, fluorocitrate, glyox, mRNA (Saccharomyces
cerevisiae), glyox (Yarrowia lipolytica)
Turnover nr:
unknown
Spec activity:
21300 μmol/min/mg (Candida tropicalis) & 22 μmol/min/mg (Yarrowia lipolytica)
Mechanism:
unknown
Location:
mitochondria
Table 11.5: Connectivity for isocitrate dehydrogenase (NAD+)
effector
organism
comment
citr
Saccharomyces cerevisiae
with constant concentrations of isocitrate, NAD+ and
Mg2+ up to 10 mM and without AMP, citrate is an
activator
mal
Saccharomyces cerevisiae
inhibitor in the absence of AMP
fum
Saccharomyces cerevisiae
inhibitor in the absence of AMP
succ
Saccharomyces cerevisiae
inhibitor in the absence of AMP
OAA
Yarrowia lipolytica
slight inhibition
NADH
Yarrowia lipolytica
complete competitive inhibition at 0.2 mM;
Ki=0.063mM
AMP
Saccharomyces cerevisiae
allosteric activation
Yarrowia lipolytica
activation
iCitr
Saccharomyces cerevisiae
Km = 0.11 mM
Yarrowia lipolytica
Km = 0.581 mM
NAD
Yarrowia lipolytica
Km = 0.136 mM
r4.5 & r4.6: iCitr + NADP => aKG + CO2 + NADPH
This reaction occurs in the mitochondria and cytosol. Nevertheless these are not parrelel reactions considering that
α-ketoglutarate can not be transported through the mitochondrial membrane.
Code:
r4.5 & r4.6
Enzyme:
isocitrate dehydrogenase (NADP)
EC number:
1.1.1.42
Reaction Brenda: iCitr + NADP = aKG + CO2 + NADPH
Reversibility:
reversible
Act & Inh:
Table 11.6
Eff. NAS:
glyox (Aspergillus cerevisiae)
Turnover nr:
unknown
Spec activity:
14.4 μmol/min/mg (Aspergillus niger), 0.24-0.3 (enzyme extract of Yarrowia lipolytica)
Mechanism:
(1) first order in regard to iCitr with competitive inhibition by aKG
(2) first order in regard to iCitr, NADP
Location:
mitochondria (r4.5) and cytosol (r4.6)
Table 11.6: Connectivity for isocitrate dehydrogenase (NADP)
effector
organism
comment
aKG
Aspergillus niger
substrate inhibition
Saccharomyces cerevisiae
0.2<Km<1.92 mM [1, 4]
- 27 -
ATP
citr
NADPH
iCitr
NADP
Aspergillus niger
Aspergillus niger
Aspergillus niger
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
inhibitor
inhibitor
inhibitor
0.01<Km<0.04 mM
0.03<Km<0.22 mM[1, 4]
0.02<Km<0.03 mM[1, 4]
r4.7: aKG + HCoA + NAD => CO2 + NADH + succCoA
Three enzymes are involved in the conversion of aKG to succCoA, which will be discussed in r4.7a-c. The most
likely inhibitors in Aspergillus niger are OAA and NADH and as a result trigger citrate accumulation in the
mitochondria [17]. Ammonia is inhibiting at high concentrations (>20 mM). For baker’s yeast cysteine and ADP
have been shown to influence the activity of oxoglutarate dehydrogenase. However cysteine is present in the
mitochondria according to the stoichiometric model and thus its influence will be neglected.
Code:
r4.7a
Enzyme:
oxoglutarate dehydrogenase (succinyl-transferring)
EC number:
1.2.4.2
Reaction KEGG: aKG + lipoamide => S-Succinyldihydrolipoamide + CO2
Reversibility:
irreversible
Act & Inh:
Table 11.7
Eff. NAS:
glyox (Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
mitochondria
Table 11.7: Connectivity for oxoglutarate dehydrogenase
effector
organism
comment
cys
Saccharomyces cerevisiae
activates aKG complex
ADP
Saccharomyces cerevisiae
stimulates
aKG
Saccharomyces cerevisiae
Km = 0.15 mM
Aspergillus niger
Km = 0.4 mM
NADH
Aspergillus niger
2-oxoglutarate dehydrogenase complex
NH4
Aspergillus niger
70% inhibition at 50 mM
OAA
Aspergillus niger
2-oxoglutarate dehydrogenase complex
succ
Aspergillus niger
2-oxoglutarate dehydrogenase complex
Code:
r4.7b
Enzyme:
dihydrolipoamide S-succinyltransferase
EC number:
2.3.1.61
Reaction KEGG: S-Succinyldihydrolipoamide + HCoA => succCoA + Dihydrolipoamide
Reversibility:
reversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
mitochondria
Code:
r4.7c
Enzyme:
dihydrolipoamide dehydrogenase
EC number:
1.8.1.4
Reaction KEGG: dihydrolipoamide + NAD => NADH + lipoamide
Reversibility:
reversible
- 28 -
Act & Inh:
Eff. NAS:
Turnover nr:
Spec activity:
Mechanism:
Location:
Table 11.8
dihydrolipoamide (Saccharomyces cerevisiae)
unknown
843 μmol/min/mg (Saccharomyces cerevisiae), 176.8 μmol/min/mg (Neurospora crassa)
unknown
mitochondria
Table 11.8: Connectivity for dihydrolipoamide dehydrogenase
effector
organism
comment
NADH
Saccharomyces cerevisiae
competitive inhibitor; Ki = 0.031 mM
Candida krusei
inhibitor
NAD
Saccharomyces cerevisiae
activator, Km = 0.15 mM
The sum of the partial reactions is:
aKG + HCoA + NAD => CO2 + NADH + succCoA
r4.8: ADP + Pi + succCoA => ATP + HCoA + succ
Code:
r4.8
Enzyme:
succinate-CoA ligase (GDP-forming)
EC number:
6.2.1.4
Reaction Brenda: ADP + Pi + succCoA => ATP + HCoA + succ
Reversibility:
reversible
Act & Inh:
none found
Eff. NAS:
none found
Spec activity:
unknown
Mechanism:
unknown
Location:
mitochondria
r4.9: FAD + succ => FADH2 + fum
Code:
r4.9
Enzyme:
succinate dehydrogenase
EC number:
1.3.99.1
Reaction Brenda: FAD + succ => FADH2 + fum
Reversibility:
reversible
Act & Inh:
none found in Brenda database
Eff. NAS:
none found in Brenda database
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
mitochondria
r4.10: fum + H2O => mal
Code:
r4.10
Enzyme:
4.2.1.2
EC number:
fumarate hydratase
Reaction Brenda: fum + H2O => mal
Reversibility:
reversible
Act & Inh:
Table 11.9
Eff. NAS:
sulfhydryl reagents (Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
1150 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
mitochondria
- 29 -
Table 11.9: Connectivity for fumarate hydratase
effector
organism
comment
Ac
Saccharomyces cerevisiae
activator at 0.2 mM
r4.11: mal + NAD + H => NADH + OAA
Code:
r4.11
Enzyme:
malate dehydrogenase
EC number:
1.1.1.37
Reaction Brenda: mal + NAD => OAA + NADH + H
Reversibility:
reversible
Act & Inh:
Table 11.10
Eff. NAS:
none found in the Brenda database
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
first order in NAD, mal or NADH [4]
Location:
mitochondria
Table 11.10: Connectivity for malate dehydrogenase
effector
organism
comment
ATP
Aspergillus niger
inhibition
mal
Saccharomyces cerevisiae
0.014<Km<0.28 mM
OAA
Aspergillus niger
product inhibition
Saccharomyces cerevisiae
0.0094<Km<0.023; Km = 75 mM [4]
NAD
Saccharomyces cerevisiae
0.11<Km<0.3 mM [1]; 0.055<Km<0.12 mM [4]
Aspergillus niger
Km = 0.083 mM
NADH
Saccharomyces cerevisiae
0.01<Km<0.03 mM [1]; 95<Km<134 mM [4]
Aspergillus niger
Km = 0.14 mM
12 Anaplerotic pathways
The stoichiometric model for a glucose grown culture of Penicillium chrysogenum excludes the glyoxylate synthesis
(r5.4) and it’s degradation to malate (r5.5).
r5.1: ATP + CO2 + H2O + pyr => ADP + 2 H + OAA + Pi
Distinct regulatory sites on the Aspergilus nidulans enzyme are occupied by L-asp and αKG. If these compounds are
present, the enzyme activitity is enhanced by AcCoA, whereas no activation occurs when these dicarboxylic acids
are absent. The activity on the Saccharomyces cerevisiae enzyme can be enhanced by long-chain acyl-CoA
derivatives, such as palmitoyl-CoA.
Pyruvate carboxylase is influenced by feedback inhibition of aKG and asp because oxaloacetate is a substrate in the
biosynthesis of aspartate (r11.16). Furthermore pyruvate carboxylase is inhibited by AcCoA by feedback due to the
biosynthesis of AcCoA and oxaloacetate in reaction r5.2.
Code:
r5.1
Enzyme:
pyruvate carboxylase
EC number:
6.4.1.1
Reaction Brenda: ATP + pyr + HCO3- = ADP + Pi + OAA
Reversibility:
reversible
Act & Inh:
Table 12.1
Eff. NAS:
long-chain acyl-CoA derivates (Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
(1) v = (Kcat*E)/(1+(K_k)/K)+Kcat0*E; K can be K+, K_k = 3.8 mM or AcCoA [4]
- 30 -
Location:
(2) v = Vmax/(1+Ka/[effector]) + R; the effector can be either AcCoA or K+ and the symbol R
corresponds to the residual velocity of the reaction in the absence of acetyl-CoA or K+ [11].
(3) v = (Kcat*E*A)/(Km_A+A) in which A is represents ATP, pyr or hydrogen-carbonate [4]
cytosol
Table 12.1: Connectivity for pyruvate carboxylase
effector
organism
comment
aKG
Saccharomyces cerevisiae
inhibitor
Aspergillus nidulans
competitive to pyr; asp at low concentrations enhances
the inhibitory effectiveness of aKG
asp
Saccharomyces cerevisiae
allosteric activator and inhibitor
Aspergillus nidulans
enhances the inactivation by aKG
AcCoA
Saccharomyces cerevisiae
activator [0.06]; 0.06<K_k<0.29 mM [4]
Aspergillus nidulans
activates
ATP
Saccharomyces cerevisiae
0.05<Km<0.07 mM [1, 4]
carbP
Saccharomyces cerevisiae
1.7<Km<12.3 mM, ADP phosphorylation [1, 11]
HCO3
Saccharomyces cerevisiae
1.36<Km<2.3 mM [1, 4]
pyr
Saccharomyces cerevisiae
0.45<Km<0.5 mM [1, 4]
r5.2: ATP + citr + HCoA => AcCoA + ADP + OAA + Pi
Although ATP citrate synthase does not seem to be present in yeast, it does occur in Penicillium spiculisporum and
Aspergillus nidulans.
Code:
r5.2
Enzyme:
ATP citrate synthase
EC number:
2.3.3.8
Reaction Brenda: ATP + citr + HCoA => ADP + Pi + AcCoA + OAA
Reversibility:
reversible
Act & Inh:
Table 12.2
Eff. NAS:
malonyl-CoA and f26P (Aspergillus nidulans)
Turnover nr:
unknown
Spec activity:
19.6 μmol/min/mg (Aspergillus nidulans)
Mechanism:
unknown
Location:
cytosol
Table 12.2: Connectivity for ATP citrate synthase
effector
organism
comment
ADP
Aspergillus nidulans
82% inhibition at 2 mM
leu
Aspergillus nidulans
20% inhibition at 2 mM
HCoA
Penicillium spiculisporum
0.001<Km<0.003 mM
citr
Penicillium spiculisporum
Km = 0.18 mM
ATP
Penicillium spiculisporum
Km = 0.09 mM
r5.3: H + NADH + OAA => mal + NAD
Malate dehydrogenase (EC 1.1.1.37) has already been dealt with in chapter 11, reaction r4.11 which is the reserve
reaction of r5.3 although it now occurs in the cytosol.
13 Oxidative Phosphorization
r6.1: 11 H:mit + 1 NADH:mit + 0.5 O2:cyt => 10 H:cyt + 1 H2O:cyt + 1 NAD:mit
r6.2: 7 H:mit + 1 NADH:cyt + 0.5 O2:cyt => 6 H:cyt + 1 H2O:cyt + 1 NAD:cyt
r6.3: 1 FADH2:mit + 6 H:mit + 0.5 O2:cyt => 1 FAD:mit + 6 H:cyt + 1 H2O:cyt
r6.4: 1 ADP:mit + 4 H:cyt + 1 Pi:mit => 1 ATP:mit + 3 H:mit + 1 H2O:mit
- 31 -
These reactions have not been found in any of the databases.
14 Different carbon substrates
The stoichiometric model does not consider different carbon substrates expect actate.
r7.2: Ac + 2 ATP + H2O + HCoA => AcCoA + 2 ADP + H + 2 Pi
Code:
r7.2a
Enzyme:
acetate-CoA ligase
EC number:
6.2.1.1
Reaction Brenda: ATP + Ac + HCoA => AMP + PP + AcCoA
Reversibility:
reversible
Act & Inh:
Table 14.1
Eff. NAS:
allicin, bicarbonate, palmitoyl-CoA and SIR2 protein (Saccharomyces cerevisiae). However it may
be possible that long chain CoA derivates which are included in the model, may also influence the
activity of this enzyme.
Turnover nr:
unknown
Spec activity:
1.241 μmol/min/mg (Saccharomyces cerevisiae) & 24 μmol/min/mg (Penicillium chrysogenum)
Mechanism:
unknown
Location:
cytosol
With the help of the partial reactions of l1 and l2, AMP and PP can be eliminated from r7.2a to obtain reaction r7.2.
Sulfate acts on acetate-CoA ligase by feedback inhibition due to the fact that in the sulfate assimilation, see
paragraph 18.7, acetate is synthesized that subsequently inhibits the activity of actetate-CoA.
Table 14.1: Connectivity for acetate-CoA ligase
effector
organism
comment
SO4
Saccharomyces cerevisiae
inhibitor
HCO3
Saccharomyces cerevisiae
bicarbonate acts as an inhibitor
Ac
Saccharomyces cerevisiae
activator; 0.2<Km<0.5 mM
Penicillium chrysogenum
Km = 6.8 mM
AcCoA
Saccharomyces cerevisiae
Km= 1.8 mM
ATP
Saccharomyces cerevisiae
0.16<Km<1.2 mM
Penicillium chrysogenum
Km = 17 mM
HCoA
Saccharomyces cerevisiae
0.035<Km<0.238 mM
Penicillium chrysogenum
Km = 0.18 mM
15 Transfer of 1-C compounds
r8.1: gly + NAD + THF => CO2 + METHF + NADH + NH4
This reaction has not yet been found in any of the online databases.
r8.2: H + METHF + NADH => MYTHF + NAD
Code:
r8.2
Enzyme:
methylenetetrahydrofolate reductase [NAD(P)H]
EC number:
1.5.1.20
Reaction Brenda: METHF + NAD(P)H => MYTHF + NAD(P)
Reversibility:
reversible
- 32 -
Act & Inh:
Eff. NAS:
Turnover nr:
Spec activity:
Mechanism:
Location:
Table 15.1
menadione (Saccharomyces cerevisiae),
unknown
unknown
unknown
cytosol
Table 15.1: Connectivity for methylenetetrahydrofolate reductase
effector
organism
comment
METHF
Saccharomyces cerevisiae
substrate inhibition; 0.01<Km<0.15 mM
SAM
Saccharomyces cerevisiae
inhibition, although the chimeric mutant is insensitive
NADH
Saccharomyces cerevisiae
Km = 0.003 mM
NADPH
Saccharomyces cerevisiae
0.0073<Km<0.021 mM
r8.3: ATP + 2 H2O +METHF + NAD => ADP + FTHF + 2 H + NADH + Pi
This reaction has not yet been found in any of the online databases.
r8.4: ATP + 2 H2O + met => H + 3 Pi + SAM
Code:
r8.4a
Enzyme:
methionine adenosyltransferasee
EC number:
2.5.1.6
Reaction Brenda: ATP + met + H2O => SAM + Pi + PP
Reversibility:
irreversible
Act & Inh:
Table 15.2
Eff. NAS:
alpha,beta-methylene-adenosine tetraphosphate, alpha,beta-methylene-ATP, CTP, GDP, GTP, L-2amino-4-hexynoic acid, S-trifluoromethyl-L-homocysteine, tetrapolyphosphate, tripolyphosphate
(Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
18.7 μmol/min/mg (Saccharomyces cerevisiae, isoenzyme I), 11 μmol/min/mg (Saccharomyces
cerevisiae, isoenzyme II)
Mechanism:
unknown
Location:
cytosol
Table 15.2: Connectivity for methionine adenosyltransferasee
effector
organism
comment
CTP
Saccharomyces cerevisiae
inhibitor
Pi
Saccharomyces cerevisiae
inhibitor, same for PP
UTP
Saccharomyces cerevisiae
inhibitor
r8.5: H2O + SAH => A + homcys
Due to the fact that adenosine reacts with ATP to synthesize ADP; both energy carriers are likely to influence the
activity of adenosylhomocysteinas. A relative low concentration of ADP might trigger an increase in activity of
adenosylhomocysteinas to produce more adenosine. Therefore εr8.5,ADP:cyt and εr8.5,ATP:cyt are non-zero.
Code:
r8.5
Enzyme:
adenosylhomocysteinas
EC number:
3.3.1.1
Reaction Brenda: SAH + H2O => A + homcys
Reversibility:
reversible
Act & Inh:
Table 15.3
- 33 -
Eff. NAS:
Turnover nr:
Spec activity:
Mechanism:
Location:
deoxyadenosine & uridine (Saccharomyces cerevisiae)
unknown
0.503 μmol/min/mg (Saccharomyces cerevisiae) & 0.01-0.012 μmol/min/mg (crude extract from
Candida utilis)
unknown
cytosol
Table 15.3: Connectivity for adenosylhomocysteinase
effector
organism
comment
ADP
Saccharomyces cerevisiae
inhibitor
AMP
Saccharomyces cerevisiae
inhibitor
ATP
Saccharomyces cerevisiae
inhibitor
cys
Saccharomyces cerevisiae
inhibitor
homcys
Saccharomyces cerevisiae
inhibitor
16 Transport across the plasma membrane
Based on the stoichiometric model, Pi (r9.2), glc (r9.3), SO4 (r9.4), Ac (r9.5) and NH4 (r9.20) are migrating across
the plasma membrane by proton symport. O2 (r9.6), CO2 (r9.7), PenG (r9.8), 6APA (r9.9), 8HPA (r9.10), PAA
(r9.11), OHPAA (r9.12), ExPept (r9.13), psacch (r9.14), H2O (r9.15), OPC (r9.16), iPN (r9.17) and PIO (r9.23) are
transported by means of passive diffusion.
It will be assumed that only the involved reactants are affecting the transport across the membrane.
17 Transport across the mitochondrial membrane
Several metabolites are transported across the mitochondrial membrane by proton symport, such as ctl (r10.7), Pi
(r10.5), ile (r10.10), pyr (r10.11), fum (r10.13), thr (r10.15), glu (r10.16), val (r10.17), gln (r10.18) and bIM (r10.19).
Some metabolites are transported by passive diffusion such as water (r10.3), ammonia (r10.9) and carbon dioxide
(r10.12). Furthermore a few metabolites are exchanged across the membrane, e.g.citrate for isocitrate (r10.6) and
citrate for malate (r10.14).
It will be assumed that only the involved reactants are affecting the transport across the membrane.
18 Amino acid synthesis
18.1
Glutamate pathway
r11.1: aKG + NADPH + NH4 + H => glu + NADP + H2O
Code:
r11.1
Enzyme:
glutamate dehydrogenase (NADP)
EC number:
1.4.1.4
Reaction Brenda: aKG + NH3 + NADPH + H => glu + H2O + NADP
Reversibility:
reversible (Aspergillus ochraceus) [1]
Act & Inh:
Table 18.1
Eff. NAS:
none found in Brenda database
Turnover nr:
unknown
Spec activity:
2.54 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
- 34 -
Table 18.1: Connectivity for glutamate dehydrogenase
effector
organism
comment
Pi
Saccharomyces cerevisiae
potassium phosphate
> 0.1 M at oxidative deamination, inhibition
< 0.1 M at oxidative deamination, activation
aKG
Saccharomyces cerevisiae
Km = 0.4 mM
Aspergillus ochraceus
Km = 2.41 mM
glu
Saccharomyces cerevisiae
6.3<Km<10 mM
NADP
Saccharomyces cerevisiae
0.01<Km<0.12 mM
NADPH
Saccharomyces cerevisiae
0.01<Km<0.03 mM
NH4
Saccharomyces cerevisiae
5<Km<10 mM
Aspergillus ochraceus
Km = 7.7 mM
r11.2: ATP + glu + NH4 => ADP + Pi + gln + H
Code:
r11.2
Enzyme:
glutamate-ammonia ligase
EC number:
6.3.1.2
Reaction Brenda: ATP + glu + NH4 = ADP + Pi + gln
Reversibility:
reversible
Act & Inh:
none found in Brenda database
Eff. NAS:
none found in Brenda database
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
18.2
Proline biosynthesis
r11.3: ATP + glu + 2 H + 2 NADPH => ADP + H2O + 2 NADP + Pi + pro
Code:
r11.3a
Enzyme:
glutamate 5-kinase
EC number:
2.7.2.11
Reaction Brenda: ATP + glu => ADP + glu-5P
Reversibility:
irreversible
Act & Inh:
none found in Brenda database
Eff. NAS:
none found in Brenda database
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Code:
r11.3b
Enzyme:
glutamate-5-semialdehyde dehydrogenase
EC number:
1.2.1.41
Reaction Brenda: glu-5P + NADPH + H => glu-5-semialdehyde + Pi + NADP
Reversibility:
reversible
Act & Inh:
none found in Brenda database
Eff. NAS:
none found in Brenda database
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
- 35 -
The metabolite 1-pyrroline-5-carboxylate is produced from the spontaneous conversion from L-glutamate 5semialdehyde which subsequently is converted into proline by pyrroline-5-carboxylate reductase.
L-glutamate 5-semialdehyde => 1-pyrroline-5-carboxylate + H2O
Code:
r11.3c
Enzyme:
pyrroline-5-carboxylate reductase
EC number:
1.5.1.2
Reaction Brenda: 1-pyrroline-5-carboxylate + NAD(P)H + H => pro + NAD(P)
Reversibility:
reversible
Act & Inh:
Table 18.2
Eff. NAS:
pyrroline-5-carboxylate, p-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoate),
ethylmaleimide (Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
15-280 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
and
N-
The sum of the all these intermediate reactions is:
glu + ATP + 2 NADPH => pro + ADP + Pi + 2 NADP + H2O
Table 18.2: Connectivity for pyrroline-5-carboxylate reductase
effector
organism
comment
NADPH
Saccharomyces cerevisiae
Km = 0.056 mM
18.3
Arginine biosynthesis
r11.4: 2 ATP + CO2 + gln + 2 H2O => 2 ADP + carbP + glu + 3H + Pi
Known effectors for this enzyme are UTP, UMP and IMP. However none of these metabolites are present in the
mitochondria, i.e. they are not synthesized in the mitrochondria or transported across the mitochondrial membrane.
Hence these effectors will be neglected.
Code:
r11.4
Enzyme:
carbamoyl-phosphate synthase (glutamine-hydrolysing)
EC number:
6.3.5.5
Reaction Brenda: 2 ATP + gln + CO2 + H2O => 2 ADP + Pi + glu + carbP
Reversibility:
irreversible
Act & Inh:
Table 18.3
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
509 μmol/min/mg (Neurospora crassa)
Mechanism:
unknown
Location:
mitochondria
Table 18.3: Connectivity for pyrroline-5-carboxylate reductase
effector
organism
comment
Pi
Saccharomyces cerevisiae
small activation effect
UTP
Saccharomyces cerevisiae
feed-back inhibition
UMP
Saccharomyces cerevisiae
inhibitor
IMP
Saccharomyces cerevisiae
activator
r11.5: ATP + carbP + 2 glu + NADPH => ADP + aKG + ctl + H + NADP + 2 Pi
- 36 -
The feedback regulation of arginine on acetylglutamate kinase will not be modeled considering arginine is not
present in the mitochondria according to the employed stoichiometric model.
Code:
r11.5a
Enzyme:
acetylglutamate kinase
EC number:
2.7.2.8
Reaction Brenda: ATP + N-acetyl-glu = ADP + N-acetyl-glu-5P
Reversibility:
irreversible
Act & Inh:
Table 18.4
Eff. NAS:
none included in the Brenda database
Turnover nr:
unknown
Spec activity:
7.21 μmol/min/mg (Neurospora crassa)
Mechanism:
unknown
Location:
mitochondria
Table 18.4: Connectivity for acetylglutamate kinase
effector
organism
comment
arg
Saccharomyces cerevisiae
feedback inhibition
Code:
r11.5b
Enzyme:
N-acetyl-gamma-glutamyl-phosphate reductase
EC number:
1.2.1.38
Reaction Brenda: N-acetyl-glu-5P + NADPH + H => N-acetyl-L-glu-5-semialdehyde + NADP + Pi
Reversibility:
reversible
Act & Inh:
none found in Brenda database
Eff. NAS:
none found in Brenda database
Turnover nr:
unknown
Spec activity:
0.953 μmol/min/mg (Neurospora crassa)
Mechanism:
unknown
Location:
mitochondria
Code:
r11.5c
Enzyme:
acetylornithine transaminase
EC number:
2.6.1.11
Reaction Brenda: N-acetyl-L-glu-5-semialdehyde + glu => N2-acetyl-L-ornithine + aKG
Reversibility:
reversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
mitochondria
Code:
r11.5d
Enzyme:
acetylornithine deacetylase
EC number:
2.3.1.35
Reaction Brenda: N2-acetyl-L-ornithine + glu => L-ornithine + N-acetyl--glu
Reversibility:
irreversible
Act & Inh:
Table 18.5
Eff. NAS:
the activity of the Saccharomyces cerevisiae enzyme is influenced by ornithine (Km = 1.5 mM),
acetyl-glutamate (Km = 17.1 mM) and acetyl-ornithine (Km = 1 mM) [1].
Turnover nr:
unknown
Spec activity:
22 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
ping pong bi bi; v = (Vmax*A*B)/(KmA*B+KmB*A+A*B) in which A corresponds to glutamate
and B with acetyl-ornithine [4]
Location:
mitochondria
- 37 -
Table 18.5: Connectivity for acetylornithine deacetylase
effector
organism
comment
glu
Saccharomyces cerevisiae
Km = 7.2 mM [1]
Code:
r11.5e
Enzyme:
ornithine carbamoyltransferase
EC number:
2.1.3.3
Reaction Brenda: carbP + L-ornithine => Pi + ctl
Reversibility:
irreversible
Act & Inh:
none found in Brenda database
Eff. NAS:
none found in Brenda database
Turnover nr:
unknown
Spec activity:
198.6-561 μmol/min/mg (Saccharomyces cerevisiae) & 251 μmol/min/mg (Neurospora crassa).
Mechanism:
unknown
Location:
mitochondria
The sum of all intermediates reactions therefore becomes equal to:
carbP + ATP + 2 glu + NADPH => ADP + aKG + ctl + Pi + NADP
r11.6: asp + 2 ATP + ctl + H2O => 2 ADP + arg + fum + H + 2 Pi
The biosynthesis of arginine proceeds via two partial reactions located in the cytosol. The first reaction is catalyzed
by argininosuccinate which is regulated by feedback control by arginine. Subsequently argininosuccinate lyase
catalyzes the synthesis of fumerate and arginine.
Code:
r11.6a
Enzyme:
argininosuccinate synthase
EC number:
6.3.4.5
Reaction Brenda: ATP + ctl + asp => AMP + PP + (Nomega-L-arginino)succinate
Reversibility:
reversible
Act & Inh:
Table 18.6
Eff. NAS:
argininosuccinate (Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
4.54 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
Table 18.6: Connectivity for argininosuccinate synthase
effector
organism
comment
arg
Saccharomyces cerevisiae
inhibition
Pi
Saccharomyces cerevisiae
inhibition
Code:
r11.6b
Enzyme:
argininosuccinate lyase
EC number:
4.3.2.1
Reaction Brenda: (Nomega-L-arginino)succinate => fum + arg
Reversibility:
reversible
Act & Inh:
none found in Brenda
Eff. NAS:
none found in Brenda
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
mitochondria
The sum of all intermediate reactions is equal to:
- 38 -
ATP + ctl +asp => arg + fum + AMP + PP
Reaction r11.6 can be derived by eliminating pyrophosphate and AMP with l2 and l1 respectively.
18.4
Lysine biosynthesis
Figure 18.1: Lysine biosynthesis [1]
r11.7: AcCoA + glu + H2O + NAD => aAd + CO2 + HCoA + NADH
The conversion of glutamine to aminoadipate proceeds through four sequential intermediate reactions.
Code:
r11.7a
Enzyme:
homocitrate synthase
EC number:
2.3.3.14
Reaction Brenda: AcCoA + H2O + aKG => homocitrate + HCoA
Reversibility:
irreversible
Act & Inh:
Table 18.7
Eff. NAS:
other inhibitors for homocitrate synthase in Saccharomyces cerevisiae are iodoacetic acid, 1,10phenanthroline, 2,2'-dipyridyl, p-hydroxymercuribenzoate, benzylpenicillium, L-norleucine, and
hydroxylysine.
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Table 18.7: Connectivity for homocitrate synthase
effector
organism
comment
at 50% inhibition by 0.053 mM, (at 6 mM aKG); Ki =
lys
Penicillium chrysogenum
0.008mM
80% inhibition at 10mM, no inhibition with D-lys
Saccharomyces cerevisiae
Ki = 5 mM
Candida maltose
HCoA
Penicillium chrysogenum
inhibitor
AMP
Penicillium chrysogenum
13-15% inhibition at 1 mM
ATP
Penicillium chrysogenum
activator
aKG
Penicillium chrysogenum
2.2<Km<5.5 mM
Candida maltose
Km = 0.025 mM
aAd
Candida maltose
Ki = 5.1 mM
- 39 -
Code:
r11.7b
Enzyme:
homoaconitate hydratase
EC number:
4.2.1.36
Reaction Brenda: homecitrate => but-1-ene-1,2,4-tricarboxylate + H2O
but-1-ene-1,2,4-tricarboxylate + H2O => homoisocitrate
Reversibility:
reversible
Act & Inh:
none identified
Eff. NAS:
the Saccharomyces cerevisiae enzyme is inhibited by 1,10-phenanthroline, 2,2'-dipyridyl and pchloromercuribenzoate.
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Code:
r11.7c
Enzyme:
homoisocitrate dehydrogenase
EC number:
1.1.1.87
Reaction Brenda: homoisocitrate + NAD => 2-oxoadipate + CO2 + NADH
Reversibility:
reversible
Act & Inh:
Table 18.8
Eff. NAS:
the Saccharomyces cerevisiae enzyme is influenced by 2-oxoadipate, homoisocitrate, and aketoadipate.
Turnover nr:
unknown
Spec activity:
3.8 mmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
Table 18.8: Connectivity for homoisocitrate dehydrogenase
effector
organism
comment
HCO3
Saccharomyces cerevisiae
activator, required for reductive carboxylation
NAD
Saccharomyces cerevisiae
Km = 0.33 mM
NADH
Saccharomyces cerevisiae
Km = 0.065 mM
Code:
r11.7d
Enzyme:
2-aminoadipate transaminase
EC number:
2.6.1.39
Reaction Brenda: 2-oxoadipate + glu => aAd + aKG
Reversibility:
reversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Reaction r11.7 can be derived by taking the sum of these four partial reactions:
AcCoA + H2O + NAD + glu => HCoA + CO2 + NADH + aAd
r11.8: aAd + 2 ATP + glu + H2O + NAD + 2 NADPH => 2 ADP + aKG + H + lys + NADH + 2 NADP + 2 Pi
Code:
r11.8a
Enzyme:
L-aminoadipate-semialdehyde dehydrogenase
EC number:
1.2.1.31
Reaction Brenda: aAd + NAD(P)H + H => L-2-aAd 6-semialdehyde + NAD(P) + H2O
Reversibility:
irreversible
- 40 -
Act & Inh:
Eff. NAS:
Turnover nr:
Spec activity:
Mechanism:
Location:
Table 18.9
EDTA (Penicillium chrysogenum), S-(beta-Aminoethyl)-L-cysteine (Candida albicans)
66 s-1 (Penicillium chrysogenum), 11.2 s-1 (Saccharomyces cerevisiae)
0.0063-0.0138 μmol/min/mg (Candida maltose)
unknown
cytosol
Table 18.9: Connectivity for L-aminoadipate-semialdehyde dehydrogenase
effector
organism
comment
inhibitor
lys
Penicillium chrysogenum
at 50mM 20% inhibition
Saccharomyces cerevisiae
70% inhibition at 50 mM
Candida albicans
ATP
Penicillium chrysogenum
activates; Km = 1.3 mM
Candida maltosa
Km = 0.5 mM
glu
Candida maltosa
competitive inhibitor with respect to aAd
asp
Candida maltosa
competitive inhibitor with respect to aAd; Ki = 4.2 mM
aAd
Candida maltosa
Km = 0.2 mM
Penicillium chrysogenum
Km = 1.4 mM
NADPH
Penicillium chrysogenum
Km = 0.16 mM
Candida maltosa
Km = 0.1 mM
Saccharomyces cerevisiae
Km = 0.62 mM
Code:
r11.8b
Enzyme:
saccharopine dehydrogenase (NADP+, L-glutamate-forming)
EC number:
1.5.1.10
Reaction Brenda: L-2-aAd 6-semialdehyde + glu + NADPH + H => N6-(L-1,3-dicarboxypropyl)-L-lys
+ NADP + H2O
Reversibility:
reversible
Act & Inh:
Table 18.10
Eff. NAS:
the Saccharomyces cerevisiae enzyme is affected by 1,10-phenanthroline, 2,2'-bipyridine,
saccharopine and p-hydroxymercuribenzoate.
Turnover nr:
unknown
Spec activity:
the purified Saccharomyces cerevisiae enzyme has a specific activity of 269.4 μmol/min/mg
whereas for the crude extract the specific activity is 0.24 μmol/min/mg. The specific activity of the
Penicillium chrysogenum is 0.044 μmol/min/mg.
Mechanism:
unknown
Location:
cytosol
Table 18.10: Connectivity for saccharopine dehydrogenase
effector
organism
comment
NADP
Saccharomyces cerevisiae
Km = 0.22 mM
Code:
r11.8c
Enzyme:
saccharopine dehydrogenase (NAD+, L-lysine-forming)
EC number:
1.5.1.7
Reaction Brenda: N6-(L-1,3-dicarboxypropyl)-L-lysine + NAD + H2O => lys + aKG + NADH + H
Reversibility:
reversible
Act & Inh:
Table 18.11
Eff. NAS:
N6-(L-1,3-dicarboxypropyl)-L-lysine effecrs the activity (Km = 1.67 mM) of the Saccharomyces
cerevisiae enzyme. Other inhibitors for this enzyme are L-ornithine, L-norleucine, L-isoleucine
and hydrophobic amino acids with 5 or 6 carbon atoms. Dicarboxylic amino acids, aspartate,
glutamate, 2-aminoadipate, pyridoxal 5'-phosphate don’t show any inhibitory effect.
Turnover nr:
18.8 s-1 (Saccharomyces cerevisiae)
- 41 -
Spec activity:
Mechanism:
Location:
24.6-106 μmol/min/mg for the purified Saccharomyces cerevisiae enzyme whereas the specific
activity of the crude extract is equal to 0.09-0.1 μmol/min/mg. The specific activity of a crude
extract of Penicillium chrysogenum is 0.381-0.571 μmol/min/mg.
unknown
cytosol
Table 18.11: Connectivity for saccharopine dehydrogenase
effector
organism
comment
lys
Saccharomyces cerevisiae
inhibitor
aKG
Saccharomyces cerevisiae
Km = 0.44 mM
Candida maltosa
Km = 0.66 mM
leu
Saccharomyces cerevisiae
activates
NADH
Saccharomyces cerevisiae
inhibition may occur; Km = 0.46 mM
Candida maltosa
Km = 0.23 mM
The sum of these reactions is therefore:
aAd + 2 NADPH + glu + NAD + H => lys + aKG + NADH + 2 NADP + H2O
This reaction is not the same as r11.8 due to the absence of ATP/ADP and Pi.
18.5
Serine biosynthesis
r11.9: 3PG + glu + H2O + NAD => aKG + H + NADH + Pi + ser
Three enzymes are involved in converting 3PG into serine. However for each of these enzymes no effectors have
been identified.
Code:
r11.9a
Enzyme:
phosphoglycerate dehydrogenase
EC number:
1.1.1.95
Reaction Brenda: 3PG + NAD => 3-phosphohydroxypyruvate + NADH + H
Reversibility:
reversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Code:
r11.9b
Enzyme:
phosphoserine transaminase
EC number:
2.6.1.52
Reaction Brenda: glu + 3-phosphohydroxypyruvate => L-phosphoserine + aKG
Reversibility:
reversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Code:
r11.9c
Enzyme:
phosphoserine phosphatase
EC number:
3.1.3.3
Reaction Brenda: L-phosphoserine + H2O => ser + Pi
Reversibility:
irreversible
- 42 -
Act & Inh:
Eff. NAS:
Turnover nr:
Spec activity:
Mechanism:
Location:
none found
none found
unknown
unknown
unknown
cytosol
The sum of all intermediate reactions is therefore
3PG + glu + NAD + H2O => aKG + ser + NADH + Pi + H
18.6
Glycine biosynthesis
r11.10: ser:cyt + THF:cyt => gly:cyt + H2O:cyt + METHF:cyt
Code:
r11.10
Enzyme:
glycine hydroxymethyltransferase
EC number:
2.1.2.1
Reaction Brenda: ser + THF => gly + METHF + H2O
Reversibility:
reversible
Act & Inh:
Table 18.12
Eff. NAS:
4-chloro-L-threonine,
beta-trifluoroallothreonine,
beta-trifluorothreonine
,
substituted
hydroxylamine derivates, sulfonyl fluoride triazine derivates & thiosemicarbazide (Saccharomyces
cerevisiae)
Turnover nr:
unkwown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Table 18.12: Connectivity for glycine hydroxymethyltransferase
effector
organism
comment
met
Saccharomyces cerevisiae
inhibitor
SAM
Saccharomyces cerevisiae
inhibitor
ser
Saccharomyces cerevisiae
0.15<Km<0.9
18.7
Sulfur metabolism
Figure 18.2: Sulfur assimilation [3]
- 43 -
r11.11: 2 ATP + 3 H + H2O + SO4 => ADP + PAPS + 2 Pi
Code:
r11.11a
Enzyme:
sulfate adenylyltransferase
EC number:
2.7.7.4
Reaction Brenda: ATP + SO4 => PP + APS
Reversibility:
reversible
Act & Inh:
Table 18.13
Eff. NAS:
the Penicillium chrysogenum enzyme is effected by 3'-phosphoadenosine-5'-phosphate (PAP) and
adenosine 5'-phosphosulfate (APS). The latter compound is also identified as a strong inhibitor for
Saccharomyces cerevisiae and Aspergillus nidulans.
Turnover nr:
unknown
Spec activity:
0.69-140 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
(1) sequential reaction mechanism in which ATP and sulfate bind before the product is released
[14]
(2) random sequence for the forward reaction and slight feedback inhibition of APS [15]
Location:
cytosol
Table 18.13: Connectivity for sulfate adenylyltransferase
effector
organism
comment
AMP
Penicillium chrysogenum
inhibitor; Ki = 0.55 mM
H2S
Saccharomyces cerevisiae
at 4 mM, 65% inhibition
met
Saccharomyces cerevisiae
at 2 mM, slightly inhibited
cys
Saccharomyces cerevisiae
at 2 mM, slightly inhibited
ADP
Saccharomyces cerevisiae
inhibitor
ATP
Saccharomyces cerevisiae
inhibitor
0.36< Km < 0.55 mM [1]; 0.29< Km<3.6 mM; Ki = 1.4
SO4
Penicillium chrysogenum
mM [4]
Km = 0.55 mM
Penicillium duponti
Pi
Penicillium chrysogenum
PP; 0.004<Km<0.008 mM [1]; 0.009.2<Km<0.025 mM
[4]
PAPS
Penicillium chrysogenum
inhibitor; 8E-5<Km<0.0004 mM
Penicillium duponti
strong inhibitor
Code:
r11.11b
Enzyme:
adenylyl-sulfate kinase
EC number:
2.7.1.25
Reaction Brenda: ATP + APS = ADP + PAPS
Reversibility:
reversible
Act & Inh:
Table 18.14
Eff. NAS:
the Saccharomyces cerevisiae enzyme is inhibited by 2,4,6-trinitrobenzene sulfonate, 2,6dichlorophenol indophenol, bromosuccinimide and dehydroascorbate, mercuriphenylacetate
whereas dithiothreitol, glutathione and thioredoxin are identified as activators. APS is known to
affect the Penicillium chrysogenum enzyme.
Turnover nr:
unknown
Spec activity:
0.208-0.843 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
compulsory ordered mechanism in which ATP binds before APS, and PAPS leaves before ADP
[16]
Location:
cytosol
Table 18.14: Connectivity for adenylyl-sulfate kinase
effector
organism
comment
PAPS
Penicillium chrysogenum
product inhibition
APS
Penicillium chrysogenum
substrate inhibition
ATP
Penicillium chrysogenum
inhibited by free ATP, i.e. in excess of total Mg2+
- 44 -
AS
Penicillium chrysogenum
FAD
Saccharomyces cerevisiae
at high salt activates at high APS conc., whereas at low
APS conc. inhibition occurs.
inhibitor
The total reaction therefore becomes:
2 ATP + SO4 => ADP + PP + PAPS
Elimination of pyrophosphate by reaction l2 yields r 11.11.
r11.12: H + 4 NADPH + PAPS => ADP + 3 H2O + H2S + 4 NADP
Code:
r11.12a
Enzyme:
phosphoadenosine phosphosulfate reductase
EC number:
1.8.1.- (no EC number assigned)
Reaction Brenda: PAPS + NADPH => sulfite + NADP + PAP
Reversibility:
unknown
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Code:
r11.12b
Enzyme:
Sulfite reductase (NADPH)
EC number:
1.8.1.2
Reaction Brenda: sulfite + 3 NADPH + 3 H+ => H2S + 3 NADP + 3 H2O
Reversibility:
reversible
Act & Inh:
Table 18.15
Eff. NAS:
8-hydroxyquinoline, mepacrine, o-phenanthroline, p-chloromercuribenzoate (Saccharomyces
cerevisiae)
Turnover nr:
unknown
Spec activity:
1.85 μmol/min/mg (Saccharomyces cerevisiae), 52 μmol/min/mg (Saccharomyces bayanus)
Mechanism:
ping-pong mechanism (Saccharomyces cerevisiae)
Location:
cytosol
Table 18.15: Connectivity for sulfite reductase
effector
organism
comment
NADP
Saccharomyces cerevisiae
competitive to NADPH, noncompetitive to sulfite
H2S
Saccharomyces cerevisiae
inhibitor
FAD
Saccharomyces cerevisiae
0.08<Km<0.083 mM
NADPH
Saccharomyces cerevisiae
0.01<Km<0.021 mM
The total reaction will therefore be:
PAPS + 4 NADPH + 3H+ => H2S + 4 NADP + 3H2O + PAP
This reaction is dissimilar to r11.12 in regard to PAP. However reaction r11.12 is lumped with the ATP hydrolyses
(l1 & r15.1) and the reactions catalyzed by adenylate kinase (l2) and 3’(2’)5’-biphosphate nucleotidase (l3).
r11.13: AcCoA + homser => AcHomser + HCoA
Cysteine, SAM and SAH are all weak inhibitor and their influence will be neglected. Methionine does not show any
inhibitory behaviour although it represses the enzyme expression.
Code:
r11.13
Enzyme:
homoserine O-acetyltransferase
EC number:
2.3.1.31
Reaction Brenda: AcCoA + homser = HCoA + AcHomser
- 45 -
Reversibility:
Act & Inh:
Eff. NAS:
Turnover nr:
Spec activity:
Mechanism:
Location:
irreversible
Table 18.16
DL-penicillamine, O-acetyl-L-serine, O-phospho-L-homoserine, O-succinyl-L-homoserine are
identified to inhibit the Saccharomyces cerevisiae enzyme.
unknown
67.4 μmol/min/mg (Saccaromyces cerevisiae) and 0.0036-3.6 μmol/min/mg (Neurospora crassa)
ping-pong mechanism (Saccaromyces cerevisiae)
cytosol
Table 18.16: Connectivity for O-acetylhomoserine aminocarboxypropyltransferase
effector
organism
comment
cys
Saccharomyces cerevisiae
weak inhibitor
homcys
Saccharomyces cerevisiae
inhibitor
met
Saccharomyces cerevisiae
enzyme expression repression, no inhibition
Achomser
Saccharomyces cerevisiae
product inhibition
SAM
Saccharomyces cerevisiae
weak inhibition
SAH
Saccharomyces cerevisiae
weak inhibition
r11.14: AcHomser + H2S => Ac + H + homcys
This enzyme is regulated by feedback control by methionine which is synthesis from homcys (r11.20).
Code:
r11.14
Enzyme:
O-acetylhomoserine aminocarboxypropyltransferase
EC number:
2.5.1.49
Reaction Brenda: AcHomser + H2S => homcys + Ac
Reversibility:
irreversible
Act & Inh:
Table 18.17
Eff. NAS:
the Saccharomyces cerevisiae enzyme is affected by hydroxylamine hydrochloride, Lpenicillamine, O-acetyl-L-serine, O-succinyl-DL-homoserine, phenylhydrazine, pyridoxal and
semicarbazide. The Aspergillus nidulans enzyme is affected by phenylhydrazine, hydroxylamine
hydrochloride and DL-C-propagylglycine
Turnover nr:
unknown
Spec activity:
4.4-8.96 μmol/min/mg (Aspergillus nidulans), 4-7.7 μmol/min/mg (Neurospora crassa), 6.86
μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
Table 18.17: Connectivity for O-acetylhomoserine aminocarboxypropyltransferase
effector
organism
comment
homser
Saccharomyces cerevisiae
competitive inhibition
met
Saccharomyces cerevisiae
competitive inhibition
Aspergillus nidulans
20% inhibition
AcHomser
Saccharomyces cerevisiae
4.1<Km<9 mM
r11.15: AcCoA + H2S + ser => Ac + cys + H + HCoA
Code:
r11.15a
Enzyme:
serine O-acetyltransferase
EC number:
2.3.1.30
Reaction Brenda: ser + AcCoA => HCoA + Acser
Reversibility:
irreversible
- 46 -
Act & Inh:
Eff. NAS:
Turnover nr:
Spec activity:
Mechanism:
Location:
none found
none found
unknown
unknown
unknown
cytosol
Code:
r11.15b
Enzyme:
cysteine synthase
EC number:
2.5.1.47
Reaction Brenda: Acser + H2S => cys + Ac
Reversibility:
irreversible
Act & Inh:
Table 18.18
Eff. NAS:
pyridoxal, pyrodoxal hydrochloride and acetyl-serine are affecting the Saccharomyces cerevisiae
enzyme
Turnover nr:
unknown
Spec activity:
0.0283-1.02 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
mechanism similar to those of other pyridoxal enzymes (Saccharomyces cerevisiae)
Location:
cytosol
Table 18.18: Connectivity for cysteine synthase
effector
organism
comment
AcHomser
Saccharomyces cerevisiae
Km=6.67 mM
Reaction r11.15 can be derived by taking the sum of r11.15a-b.
18.8
Alanine & aspartate metabolism
r11.16: glu + OAA => aKG + asp
Code:
r11.16
Enzyme:
aspartate transaminase
EC number:
2.6.1.1
Reaction Brenda: OAA + glu => aKG + asp
Reversibility:
reversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
r11.17: asp + 2 ATP + H2O + NH4 => 2 ADP + asn + 2 H + 2 Pi
Code:
r11.17
Enzyme:
aspartate-ammonia ligase
EC number:
6.3.1.1
Reaction Brenda: ATP + L-aspartate + NH3 => AMP + asn + PP
Reversibility:
irreversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
- 47 -
r11.22: glu + pyr => aKG + ala
Code:
r11.22
Enzyme:
alanine transaminase
EC number:
2.6.1.2
Reaction Brenda: pyr + glu => aKG + ala
Reversibility:
reversible
Act & Inh:
Table 18.19
Eff. NAS:
aminooxyacetate, hydroxylamine and p-hydroxymercuribenzoate (Candida maltose)
Turnover nr:
unknown
Spec activity:
594 μmol/min/mg forward reaction and 156 μmol/min/mg reserve reaction (Candida maltose)
Mechanism:
unknown
Location:
cytosol
Table 18.19: Connectivity for alanine transaminase
effector
organism
comment
aKG
Candida maltose
Km = 0.37 mM
ala
Candida maltose
Km = 2.6 mM
glu
Candida maltose
Km = 6.3 mM
pyr
Candida maltose
Km = 0.16 mM
18.9
Threonine biosynthesis
Figure 18.3: Threonine biosynthesis (E.Coli) [3]
r11.18: asp + ATP + 2 H + 2 NADPH => ADP + homser + 2 NADP + Pi
Code:
r11.18a
Enzyme:
aspartate kinase
EC number:
2.7.2.4
Reaction Brenda: ATP + asp => ADP + 4P-asp
Reversibility:
reversible
Act & Inh:
Table 18.20
Eff. NAS:
none found
- 48 -
Turnover nr:
Spec activity:
Mechanism:
Location:
unknown
8.2 μmol/min/mg (Saccharomyces cerevisiae)
(1) v = (E*kcat*substrate)/(Km+substrate) [4]
(2) v = A-D/(1+ (I/IC50)S) + D; A and D is minium and maximum response plateau respectively, S
is the slope factor and I is concentration of inhibitor [12].
(3) mixed inhibition v = Vmax*S/(Km(1+I/Kis)+S(1+I/Kic)); I is inhibitor and S is substrate [12].
(4) uncompetitive inhibition v = Vmax*S/(Km + S(1+I/Ki’)) [12]
cytosol
Table 18.20: Connectivity for aspartate kinase
effector
organism
comment
thr
Saccharomyces cerevisiae
inhibitor [1]; 2.7<IC50<38 mM, 7.6<Kic<60 mM,
1.9<Kis<14 mm, Ki’=28 mM, [4, 12]
ATP
Saccharomyces cerevisiae
5.4<Km<10.4 mM [1]; 0.26<Km<3.36 mM [4, 12]
asp
Saccharomyces cerevisiae
11.3<Km<12.4 mM [1]; 1.63<Km<11.9 mM [4, 12]
Code:
r11.18b
Enzyme:
aspartate-semialdehyde dehydrogenase
EC number:
1.2.1.11
Reaction Brenda: 4P-asp + NADPH + H => asp 4-semialdehyde + Pi + NADP
Reversibility:
reversible
Act & Inh:
Table 18.21
Eff. NAS:
iodoacetate, iodoacetamide, 4P-asp, N-ethylmaleimide and asp 4-semialdehyde affect the
Saccharomyces cerevisiae enzyme.
Turnover nr:
unknown
Spec activity:
73 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
Table 18.21: Connectivity for aspartate-semialdehyde dehydrogenase
effector
organism
comment
CO2
Saccharomyces cerevisiae
H2CO3, enhances 4-5x activity
met
Saccharomyces cerevisiae
inhibitor
thr
Saccharomyces cerevisiae
inhibitor
homcys
Saccharomyces cerevisiae
inhibitor in the reserve reaction; Ki = 10 mM
ATP
Saccharomyces cerevisiae
inhibit at 0oC and 2mM dithiothreitol
cys
Saccharomyces cerevisiae
inhibitor in the reserve reaction
Pi
Saccharomyces cerevisiae
potassium phosphate; Km = 1.4 mM
NADPH
Saccharomyces cerevisiae
Km = 0.083 mM
NADP
Saccharomyces cerevisiae
Km = 0.036 mM
Code:
r11.18c
Enzyme:
homoserine dehydrogenase
EC number:
1.1.1.3
Reaction Brenda: asp 4-semialdehyde + NAD(P)H + H => homser + NAD(P)
Reversibility:
reversible
Act & Inh:
Table 18.22
Eff. NAS:
4,4'-thiobis-(di)methylethyl-phenol derivates, bis(4-chlorophenyl)ethyloxiranyl-silane, H-(1,2,4triazol-3-yl)-DL-alanine, 4-(1-methylheptyl)-1,3-benzenediol (Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
23.7-51 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
Table 18.22: Connectivity for homoserine dehydrogenase
- 49 -
effector
thr
organism
Saccharomyces cerevisiae
met
Saccharomyces cerevisiae
comment
weakly inhibits reverse reaction, no effect on forward
reaction.
weakly inhibit reserve reaction, no effect in forward
reaction.
Summing these intermediate reactions will yield a similar reaction as r11.18.
asp + ATP + 2 NADPH + 2H => homser + ADP + 2 NADP + Pi
r11.19: ATP + H2O + homser => ADP + H + Pi + thr
Code:
r11.19a
Enzyme:
homoserine kinase
EC number:
2.7.1.39
Reaction Brenda: ATP + homser => ADP + O-phospho-L-homoserine
Reversibility:
reversible
Act & Inh:
Table 18.23
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
3.1 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
Table 18.23: Connectivity for homoserine kinase
effector
organism
comment
homser
Saccharomyces cerevisiae
Km = 0.25 mM; Ki = 2 mM
thr
Saccharomyces cerevisiae
inhibitor
ATP
Saccharomyces cerevisiae
Km = 0.6 mM
Code:
r11.19b
Enzyme:
threonine synthase
EC number:
4.2.3.1
Reaction Brenda: O-phospho-L-homoserine + H2O => thr + Pi
Reversibility:
irreversible
Act & Inh:
Table 18.24
Eff. NAS:
phospho-threonine, NH2OH (Neurospora crassa)
Turnover nr:
unknown
Spec activity:
1.73 μmol/min/mg (Neurospora crassa)
Mechanism:
unknown
Location:
cytosol
Table 18.24: Connectivity for threonine synthase
effector
organism
comment
cys
Neurospora crassa
85% inhibition at 35 mM
Pi
Neurospora crassa
40% inhibition at 50 mM
Summing these reactions will yield r11.19
ATP + H2O + homser => ADP + Pi + thr
18.10 Methionine biosynthesis
r11.20: METHF + homcys => THF + met
Code:
Enzyme:
r11.20
methionine synthase
- 50 -
EC number:
2.1.1.13
Reaction Brenda: METHF + homcys = THF + met
Reversibility:
irreversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
18.11 Leucine, isoleucine & valine metabolism
Figure 18.4: Isoleucine synthesis [3]
Figure 18.5: Valine synthesis [3]
- 51 -
Figure 18.6: Leucine biosynthesis [3]
r11.21: glu + 2 H + NADPH + pyr + thr => aKG + CO2 + H2O + ile + NADP + NH4
Code:
r11.21a
Enzyme:
threonine ammonia-lyase
EC number:
4.3.1.19
Reaction Brenda: thr => 2-oxobutanoate + NH3
Reversibility:
reversible
Act & Inh:
Table 18.25
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
mitochondria
Table 18.25: Connectivity for threonine ammonia-lyase
effector
organism
comment
Pi
Saccharomyces cerevisiae
activator
Candida maltose
activator
val
Candida maltose
activator
thr
Candida maltose
2.5<Km< 4 mM
ile
Candida maltose
Ki = 0.14 mM
Code:
r11.21b
Enzyme:
acetolactate synthase
EC number:
4.1.3.18 (transferred to 2.2.1.6 which is discussed in r11.21b2)
Reaction Brenda: pyr + 2-oxobutanoate => 2-aceto-2-hydroxy-butyrate + CO2
Reversibility:
irreversible
Act & Inh:
none found
Eff. NAS:
2-oxobutanoate, PCMB (Neurospora crassa)
Turnover nr:
unknown
Spec activity:
0.4075 μmol/min/mg (Neurospora crassa)
Mechanism:
unknown
Location:
mitochondria
Table 18.26: Connectivity for acetolactate synthase
effector
organism
comment
ile
Schizosaccharomyces p.
noncompetitive inhibitor
val
Neurospora crassa
noncompetitive inhibitor
Schizosaccharomyces p.
noncompetitive inhibitor, feed-back inhibition
pyr
Neurospora crassa
3.2<Km<17 mM
- 52 -
Saccharomyces cerevisiae
Km = 3.93 mM
Code:
r11.21b2
Enzyme:
acetolactate synthase
EC number:
2.2.1.6
Reaction Brenda: pyr + 2-oxobutanoate => 2-aceto-2-hydroxy-butyrate + CO2
Reversibility:
reversible
Act & Inh:
Table 18.27
Eff. NAS:
requires thiamine diphosphate as cofactor (Saccharomyces cerevisiae). PCMB and 2-oxobutanoate
inhibit the activity of the Neurospora crassa enzyme.
Turnover nr:
unknown
Spec activity:
0.4075 μmol/min/mg (Neurospora crassa)
Mechanism:
unknown
Location:
mitochondria
Table 18.27: Connectivity for acetolactate synthase
effector
organism
comment
pyr
Saccharomyces cerevisiae
Km = 3.93 mM
Neurospora crassa
3.2<Km<17 mM
FAD
Saccharomyces cerevisiae
Km = 0.0003 mM
val
Neurospora crassa
noncompetitive pH dependent inhibitor
ile
Schizosaccharomyces
inhibitor
pombe
Code:
r11.21c
Enzyme:
ketol-acid reductoisomerase
EC number:
1.1.1.86
Reaction Brenda: NADPH + H + 2-aceto-2-hydroxybutyrate => NADP + (R)-2,3-dihydroxy-3methylvalerate
Reversibility:
reversible
Act & Inh:
Table 18.28
Eff. NAS:
the Neurospora crassa enzyme is influenced by 2,3-dihydroxy-3-methylvalerate and
2-acetolacetate.
Turnover nr:
unknown
Spec activity:
18.5 μmol/min/mg (Neurospora crassa)
Mechanism:
unknown
Location:
mitochondria
Ketol-acid reductoisomerase can employ NAD or NADP as acceptors.
Table 18.28: Connectivity for ketol-acid reductoisomerase
effector
organism
comment
NADP
Neurospora crassa
inhibitor
NADH
Neurospora crassa
inhibitor
Code:
r11.21d
Enzyme:
dihydroxy-acid dehydratase
EC number:
4.2.1.9
Reaction Brenda:(R)-2,3-dihydroxy-3-methylvalerate => 2 keto-3-methyl-3-methylvalerate + H2O
Reversibility:
reversible
Act & Inh:
none found
Eff. NAS:
the Neurospora crassa enzyme is influenced by 2,3-dihydroxy-3-methylvalerate and (R)-2,3dihydroxy-3-methylbutanoate.
Turnover nr:
unknown
Spec activity:
2.15-10.13 μmol/min/mg (Neurospora crassa)
- 53 -
Mechanism:
Location:
unknown
mitochondria
Code:
r11.21e
Enzyme:
branched-chain-amino-acid transaminase
EC number:
2.6.1.42
Reaction Brenda: 2 keto-3-methyl-3-methylvalerate + glu => ile + aKG
Reversibility:
reversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
48.8 μmol/min/mg for the mitochiondrial Neurospora crassa enzyme. It’s cytoplasmic counterpart
has a specific activity of 25 μmol/min/mg.
Mechanism:
unknown
Location:
mitochondria
The sum of these reactions which will yield r11.21.
thr + pyr + NADPH + H + glu => NH3 + CO2 + NADP + H2O + ile + aKG
r11.23: 2 H + NADPH + 2 pyr => aKI + CO2 + H2O + NADP
The involved enzymes have already been discussed in reaction r11.21. The valine biosynthesis initiates by
converting pyruvate into 2-acetolacetate also known as 2-aceto-lactate, which is catalysed by threonine ammonialyase (EC 4.3.1.19).
2 pyr => 2-aceto-lactate + CO2
Subsequently the enzymes ketol-acid reductoisomerase (EC 1.1.1.86) and dihydroxy-acid dehydratase (EC 4.2.1.9)
catalyze respectively:
NADPH + 2-aceto-lactate => NADP + 2,3-hydroxy-isovalerate
2,3-hydroxy-isovalerate => aKI + H2O
The sum of the partial reactions are:
2 pyr + NADPH => CO2 + NADP + H2O + aKI
r11.24: aKI + glu => val + aKG
Branched-chain-amino-acid transaminase (EC 2.6.1.42) has already been discussed for reaction r11.21 in which it
catalyzes the final step of the synthesis of isoleucine. However it also catalyzes the final step of the valine synthesis.
The reaction is as follows
aKI + glu => val + aKG
r11.25: AcCoA + aKI + H2O => bIM + H + HCoA
Code:
r11.25a
Enzyme:
2-isopropylmalate synthase
EC number:
2.3.3.13
Reaction Brenda: aKI + AcCoA + H2O => 3-carboxy-3-hydroxy-isocaproate + HCoA
Reversibility:
irreversible
Act & Inh:
Table 18.29
Eff. NAS:
The Saccharomyces cerevisiae enzyme is inhibited by 2-oxo-isohexanoate, 2-oxopentanoate,
5',5',5'-trifluoroleucine, D-leucine amide, EDTA, 2-oxobutanoate, 2-oxo-3-methylbutanoate.
Turnover nr:
unknown
Spec activity:
1.58-7.1 μmol/min/mg (Saccharomyces cerevisiae).
Mechanism:
unknown
Location:
mitochondria
3-carboxy-3-hydroxy-isocaproate is also known as α-isopropylmalate. The EC number 4.1.3.12 in Figure 18.6 is
transferred to EC 2.3.3.13.
- 54 -
Table 18.29: Connectivity for 2-isopropylmalate synthase
effector
organism
comment
leu
Saccharomyces cerevisiae
inhibitor; 0.1<Ki<1.2 mM
HCoA
Saccharomyces cerevisiae
product inhibitor; Ki = 0.07 mM
pyr
Saccharomyces cerevisiae
Km = 0.2 mM
phe
Neurospora crassa
inhibitor
val
Neurospora crassa
inhibitor
AcCoA
Neurospora crassa
0.0245<Km<0.035 mM
Code:
r11.25b
Enzyme:
3-isopropylmalate dehydratase
EC number:
4.2.1.33
Reaction Brenda: 3-carboxy-3-hydroxy-isocaproate => bIM
Reversibility:
reversible
Act & Inh:
Table 18.30
Eff. NAS:
the Saccharomyces cerevisiae enzyme is affected by 8-hydroxyquinolinesulfonate and 1,10phenanthroline, citraconate and dimethylcitraconate, whereas the Neurospora crassa enzyme is
inhibited by glutathione, mercaptoethanol, sufhydryl compounds and beta-carboxy-betahydroxyisocaproate.
Turnover nr:
unknown
Spec activity:
6.18 μmol/min/mg (Saccharomyces cerevisiae), 6.2 μmol/min/mg (Neurospora crassa)
Mechanism:
unknown
Location:
mitochondria
bIM is also known as β-isopropylmalate. 3-isopropylmalate dehydratase first catalyzes 3-carboxy-3-hydroxyisocaproate into 2-isopropylmaleate, after which it is converted into bIM. The enzyme activity is also influenced by
glycerol and (NH4)2SO4 under high ionic stengh conditions. However these conditions are not encounted in the cell
and therefore it will be assumed that their influence under normal conditions will be neglectable.
Table 18.30: Connectivity for 3-isopropylmalate dehydratase
effector
organism
comment
cys
Neurospora crassa
inhibitor
(NH4)2SO4
Saccaromyces cerevisiae
influences enzyme at high ionic strength conditions
The sums of the intermediate reactions are:
aKI + AcCoA + H2O => HCoA + bIM
r11.26: bIM + glu + NAD => aKG + CO2 + leu + NADH
Code:
r11.25
Enzyme:
3-isopropylmalate dehydrogenase
EC number:
1.1.1.85
Reaction Brenda: bIM + NAD => 2-isopropyl-3-oxosuccinate + NADH + H
Reversibility:
reversible
Act & Inh:
Table 18.31
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
18.4-19.3 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
Table 18.31: Connectivity for 3-isopropylmalate dehydrogenase
effector
organism
comment
bIM
Saccharomyces cerevisiae
0.023<Km<0.042 mM
- 55 -
NAD
Saccharomyces cerevisiae
0.054<Km<0.15 mM
Subsequently 2-isopropyl-3-oxosuccinate spontaneously converts into:
2-isopropyl-3-oxosuccinate => 2-ketoisocaproate + CO2
This is followed by biosynthesis of leucine that is catalyzed by branched-chain-amino-acid transaminase (EC
2.6.1.42), which has previously already been discussed for r11.21e.
2-ketoisocaproate + glu => aKG + leu
18.12 Phenylalanine, tyrosine and tryptophan biosynthesis
Figure 18.7: Chorismate synthesis [3]
r11.27: ATP + E4P + NADPH + 2 PEP => ADP + chor + NADP + 4 Pi
Code:
r11.27a
Enzyme:
3-deoxy-7-phosphoheptulonate synthase
EC number:
2.5.1.54 (transferred from 4.1.2.15)
Reaction Brenda: E4P + PEP + H2O => 3-deoxy-D-arabino-heptulosonate-7-phosphate + Pi
Reversibility:
irreversible
Act & Inh:
Table 18.32
Eff. NAS:
EDTA
(Saccharomyces
cerevisiae),
7-Phospho-2-dehydro-3-deoxy-D-arabino-heptonate
(Neurospora crassa)
Turnover nr:
unknown
Spec activity:
8.24 μmol/min/mg (Saccharomyces cerevisiae) & 7.7-8.1 μmol/min/mg (Neurospora crassa
purified trpI), 4.5 μmol/min/mg (Neurospora crassa partially purified pheI), 0.27 μmol/min/mg
(Neurospora crassa partially purified tyrI)
Mechanism:
unknown
Location:
cytosol
Three isoenzymes exist, the phe-sensitive isoenzyme (pheI), the tyr-sensitive isoenzyme (tyrI) and the Trp-sensitive
isozyme (trpI).
Table 18.32: Connectivity for 3-deoxy-7-phosphoheptulonate synthase
effector
organism
comment
- 56 -
phe
tyr
E4P
PEP
trp
Pi
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Neurospora crassa
Saccharomyces cerevisiae
Neurospora crassa
Neurospora crassa
0.01 (pheI) < Ki < 0.27 mM (tyrI)
Ki = 0.0009 mM (tyrI)
0.13 (pheI) < Km < 0.5 mM (tyrI)
substrate inhibition
0.018 (pheI) < Km < 0.125 mM (tyrI)
inhibitor (trpI)
non-competitive inhibitor to both PEP and E4P
Code:
r11.27b
Enzyme:
3-dehydroquinate synthase
EC number:
4.2.3.4
Reaction Brenda: 3-deoxy-D-arabino-heptulosonate-7-phosphate => 3-dehydroquinate + Pi
Reversibility:
irreversible [2], reversible [4]
Act & Inh:
Table 18.33
Eff. NAS:
the Aspergillus nidulans enzyme is influenced by EDTA, 3-deoxy-D-arabino-heptulosonate
phosphate (0.009<Km<0.028 mM), carabaphosphonate and diethyl dicarbonate, wheareas the
Neurospora crassa enzyme is influenced by EDTA, 3-deoxy-D-arabino-heptulosonic acid 7phosphate ,1,10-phenanthroline, bistrispropane and dithiothreitol
Turnover nr:
19 s-1 (Neurospora crassa)
Spec activity:
14.9 μmol/min/mg (Aspergillus nidulans)
Mechanism:
sequential ordered multistep [4]
Location:
cytosol
Table 18.33: Connectivity for 3-deoxy-7-phosphoheptulonate synthase
effector
organism
comment
NADH
Neurospora crassa
inhibitor
NAD
Aspergillus nidulans
Km = 0.003 mM
Code:
r11.27c
Enzyme:
3-dehydroquinate dehydratase
EC number:
4.2.1.10
Reaction Brenda: 3-dehydroquinate => 3-dehydroshikimate + H2O
Reversibility:
reversible
Act & Inh:
none found
Eff. NAS:
the Aspergillus nidulans enzyme is influenced by cyclohexanecarboxylic acid derivates, 3dehydroquinate (Km = 40 mM), diethyl dicarbonate, 3-deoxyquinic acid and 2,3-anhydroquinic
acid. The Neurospora crassa enzyme is inhibited by guanidine hydrochloride and SDS.
Turnover nr:
unknown
Spec activity:
1284 μmol/min/mg (Aspergillus nidulans), 0.096 μmol/min/mg (Neurospora crassa)
Mechanism:
sequential ordered multistep [4]
Location:
cytosol
Code:
r11.27d
Enzyme:
shikimate dehydrogenase
EC number:
1.1.1.25
Reaction Brenda: 3-dehydroshikimate + NADPH + H => shikimate + NADP
Reversibility:
reversible
Act & Inh:
none found
Eff. NAS:
3-dehydroshikimate (Km= 0.311 mM) (Aspergillus nidulans) [4]
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
sequential ordered multistep [4]
Location:
cytosol
Table 18.34: Connectivity for shikimate dehydrogenase
- 57 -
effector
NADPH
organism
Aspergillus nidulans
comment
Km = 0.0135 mM [4]
Code:
r11.27e
Enzyme:
shikimate kinase
EC number:
2.7.1.71
Reaction Brenda: ATP + shikimate => ADP + shikimate 3-phosphate
Reversibility:
irreversible
Act & Inh:
none found
Eff. NAS:
shikimate (Km = 22.3 mM) (Aspergillus nidulans) [4]
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
sequential ordered multistep [4]
Location:
cytosol
Table 18.35: Connectivity for shikimate kinase
effector
organism
comment
ATP
Aspergillus nidulans
Km = 4.8 mM [4]
Code:
r11.27f
Enzyme:
3-phosphoshikimate 1-carboxyvinyltransferase
EC number:
2.5.1.19
Reaction Brenda: PEP + 3-phosphoshikimate => Pi + 5-enolpyruvyl-shikimate 3-phosphate.
Reversibility:
reversible
Act & Inh:
Table 18.36
Eff. NAS:
the Neurospora crassa enzyme is influenced by 3-phosphoshikimate, N-phosphonomethylglycine
and 5-enolpyruvyl-shikimate 3-phosphate.
Turnover nr:
33.6 s-1 (Neurospora crassa)
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Table 18.36: Connectivity for 3-phosphoshikimate 1-carboxyvinyltransferase
effector
organism
comment
PEP
Neurospora crassa
Km = 0.0018 mM
Pi
Neurospora crassa
Km = 0.0027 mM
Code:
r11.27g
Enzyme:
chorismate synthase
EC number:
4.2.3.5
Reaction Brenda: 5-enolpyruvyl-shikimate 3-phosphate => chor + Pi
Reversibility:
irreversible
Act & Inh:
Table 18.37
Eff. NAS:
O5-(1-carboxyvinyl)-3-phosphoshikimate, riboflavin-5-phosphate (Neurospora crassa). For the
latter organism the Sabio RK database includes that the Michaelis-Menten coefficient for O5-(1carboxyvinyl)-3-phosphoshikimate is between 0.039-0.083 mM
Turnover nr:
unknown
Spec activity:
32.1 mmol/min/mg (Neurospora crassa)
Mechanism:
unknown
Location:
cytosol
Table 18.37: Connectivity for chorismate synthase
effector
organism
comment
NADPH
Neurospora crassa
Km = 0.0018 mM; required cofactor [1]
- 58 -
0.043<Km<0.28 mM [4]
The sum of the intermediate reaction steps is:
E4P + 2 PEP + NADPH + ATP => chor + NADP + ADP + 4 Pi
r11.28: chor + glu + H => aKG + CO2 + H2O + phe
Apart from the reactants, the involved enzymes for the biosynthesis of phenylalanine are influenced by feedback
control of phenylalanine, tyrosine and tryptophane.
Code:
r11.28a
Enzyme:
chorismate mutase
EC number:
5.4.99.5
Reaction Brenda: chor => prephenate
Reversibility:
reversible
Act & Inh:
Table 18.38
Eff. NAS:
caffeic acid, 3,4-dimethoxycinnamic acid derivates (Penicillium chrysogenum/duponti).
Turnover nr:
the turnover number of the wild type enzyme in baker’s yeast is 361 s-1. For Aspergillus nidulans
the turnover number is 82 s-1. In the presence of 0.005 mM trp the turnover number is enhanced to
92 s-1.
Spec activity:
8400-88500 μmol/min/mg (Aspergillus nidulans)
Mechanism:
unknown
Location:
cytosol
Table 18.38: Connectivity for chorismate mutase
effector
organism
comment
phe
Neurospora crassa
inhibitor
Penicillium duponti
inhibitor
Penicillium chrysogenum
inhibitor
tyr
Neurospora crassa
inhibitor
Penicillium duponti
inhibitor
Penicillium chrysogenum
inhibitor
Saccharomyces cerevisiae
inhibitor
trp
Neurospora crassa
activator
Penicillium duponti
activator
Penicillium chrysogenum
activator
Saccharomyces cerevisiae
activator
chor
Saccharomyces cerevisiae
0.4 < Km < 6.3 mM
Code:
r11.28b
Enzyme:
prephenate dehydratase
EC number:
4.2.1.51
Reaction Brenda: prephenate => phenylpyruvate + H2O + CO2
Reversibility:
reversible
Act & Inh:
Table 18.39
Eff. NAS:
The candida maltose enzyme is influenced by prephenate, 2-methyl-DL-tyrosine, 5fluorotryptophan.
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Subsequently the following reaction takes place:
prephenate = phenylpyruvate + H2O + CO2
Table 18.39: Connectivity for prephenate dehydratase
effector
organism
comment
- 59 -
tyr
trp
Candida Maltose
Candida Maltose
inhibitor
activation, Km = 0.56 mM
In the final reaction step, phenylpyruvate is converted into phenylalanine:
phenylpyruvate + glu => phe + aKG
Code:
r11.28c
Enzyme:
histidinol-phosphate transaminase
EC number:
2.6.1.9
Reaction Brenda: phenylpyruvate + glu => phe + aKG
Reversibility:
reversible
Act & Inh:
none found
Eff. NAS:
hydroxylamine, iodoacetate and semicarbazide (Neurospora sp.)
Turnover nr:
unknown
Spec activity:
60.3 μmol/min/mg (Neurospora crassa)
Mechanism:
unknown
Location:
cytosol
The sum of the intermediate reactions is:
chor + glu => phe + aKG + H2O + CO2
r11.29: chor + glu + NAD => aKG + CO2 + NADH + tyr
The first step in the biosynthesis of tyrosine is the conversion of chorismate to prephenate which is carried out by
chorismate mutase. This enzyme has already been elaborated in reaction r11.28.
Subsequently prephanate is converted to p-hydroxyphenylpyruvate, which is catalyzed by prephenate dehydrogenase
(EC 1.3.1.12) after which p-hydroxyphenylpyruvate is converted into tyrosine by the enzymes discussed in r11.28c.
Code:
r11.29a
Enzyme:
prephenate dehydrogenase
EC number:
1.3.1.12
Reaction Brenda: prephenate + NAD => 4-hydroxyphenylpyruvate + CO2 + NADH
Reversibility:
reversible
Act & Inh:
Table 18.40
Eff. NAS:
alpha-Methyl-D,L-tyrosine, prephenate, L-tryptophan analogues (Candida maltose)
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Table 18.40: Connectivity for prephenate dehydrogenase
effector
organism
comment
tyr
Candida Maltose
inhibitor; Ki = 5.5 mM
inhibitor
Neurospora sp.
phe
Neurospora sp.
activator
trp
Candida maltose
activator
NAD
Candida maltose
0.13 < Km < 0.15 mM
Like for r11.28, it is assumed that histidinol-phosphate transaminase (EC 2.6.1.9) will catalyze the final step of the
biosynthesis of tyrosine. The catalyzed reaction proceeds as follows:
4-hydroxyphenylpyruvate + glu => tyr + aKG
The sum of the intermediate reactions is:
chor + NAD + glu =>tyr + aKG + CO2 + NADH
r11.30: chor + gln + PPRP + ser => CO2 + GAP + glu + H + H2O + 2 Pi + pyr + trp
- 60 -
The only effector found is cysteine.
Code:
r11.30a
Enzyme:
anthranilate synthase
EC number:
4.1.3.27
Reaction Brenda: chor + gln => anthranilate + pyr + glu
Reversibility:
irreversible
Act & Inh:
none found, although many organism show trp as inhibitor
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Code:
r11.30b
Enzyme:
anthranilate phosphoribosyltransferase
EC number:
2.4.2.18
Reaction Brenda: anthranilate + PRPP => N-(5-phospho-D-ribosyl)-anthranilate + PP
Reversibility:
reversible
Act & Inh:
none found
Eff. NAS:
N-(5-phospho-D-ribosyl)-anthranilate and anthranilate (Saccharomyces cerevisiae)
Turnover nr:
2.9 s-1 (Saccharomyces cerevisiae)
Spec activity:
1.58 μmol/min/mg (Saccharomyces cerevisiae), 16 μmol/min/mg (Neurospora crassa)
Mechanism:
unknown
Location:
cytosol
Code:
r11.30c
Enzyme:
phosphoribosylanthranilate isomerase
EC number:
5.3.1.24
Reaction Brenda: N-(5-phospho-beta-D-ribosyl)anthranilate
=>
1-(2-carboxyphenylamino)-1-deoxy-Dribulose 5-phosphate.
Reversibility:
irreversible (Aspergillus nidulans)
Act & Inh:
none found
Eff. NAS:
N-(5-phospho-beta-D-ribosyl)anthranilate and reduced 1-(2-carboxyphenylamino)-1-deoxy-Dribulose 5-phosphate (Saccharomyces cerevisiae)
Turnover nr:
50-69 s-1 (Saccharomyces cerevisiae)
Spec activity:
102 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
Code:
r11.30d
Enzyme:
indole-3-glycerol-phosphate synthase
EC number:
4.1.1.48
Reaction Brenda: 1-(2-carboxyphenylamino)-1-deoxy-D-ribulose 5-phosphate => indole-3-glycerol
phosphate + CO2 + H2O
Reversibility:
reversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Subsequently indole-3-glycerol-phosphate spontaneously converts into:
indole-3-glycerol-phosphate => GAP + indole
- 61 -
Code:
r11.30e
Enzyme:
tryptophan synthase
EC number:
4.2.1.20
Reaction Brenda: indole + ser => trp + H2O
Reversibility:
reversible
Act & Inh:
Table 18.41
Eff. NAS:
indole, several indole derivates such as indoleacetic acid, indoleacrylic acid, indolebutyric acid,
indolepropionic acid, indolepyruvic acid, 5,5'-dithiobis(2-nitrobenzoate), GSH and PCMB
(Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
1.69 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
Table 18.41: Connectivity for tryptophan synthase
effector
organism
comment
cys
Saccharomyces cerevisiae
inhibitor
GAP
Saccharomyces cerevisiae
Km = 1.1 mM
ser
Saccharomyces cerevisiae
Km = 5.3 mM
The sum of these intermediates reactions is:
chor +gln + PRPP + ser => glu + pyr + PP + CO2 + 2H2O + GAP + trp
18.13 PRPP biosynthesis
r11.31: 2 ATP + Ribu5P => 2 ADP + H + PRPP
Code:
r11.31a
Enzyme:
ribose-phosphate diphosphokinase
EC number:
2.7.6.1
Reaction Brenda: Rib5P + ATP => PRPP + AMP
Reversibility:
reversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
The stoichiometric model however describes the biosynthesis of PRPP from ribulose-5-phosphate and therefore an
additional reaction is needed which is already been elaborated in the pentose phosphate pathway, i.e. reaction r2.3.
The sum of the intermediate reactions therefore becomes:
Ribu5P + ATP => PRPP + AMP
Elimination of AMP with reaction l1 will yield reaction r11.
18.14 Histidine biosynthesis
r11.32: 3 ATP + CO2 + gln + 3 H2O + 2 NAD + NADPH + NH4 + PRPP => 3 ADP + aKG + 8 H + his + 2
NADH + NADP + 6 Pi
This reaction has not been found in any of the online databases
r11.32: ATP phosphoribosyltransferase (EC 2.4.2.17)
The following reaction is catalyzed by this enzyme:
- 62 -
ATP + PRPP => 1-(5-phospho-D-ribosyl)-ATP + PP
No effectors of this enzyme in fungi are included in the Brenda database.
r11.32: Phosphoribosyl-ATP diphosphatase (EC 3.6.1.31)
This enzyme is involved the following reaction:
1-(5-phosphoribosyl)-ATP + H2O = 1-(5-phosphoribosyl)-AMP + PP
No effectors have been identified for fungi.
r11.32: Phosphoribosyl-AMP cyclohydrolase (EC 3.5.4.19)
The reaction proceeds as according to:
1-(5-phosphoribosyl)-AMP + H2O => phosphoribosylformiminoAICAR-phosphate
No effectors have been identified for fungi.
r11.32:
1-(5-phosphoribosyl)-5-[(5-phosphoribosylamino)methylideneamino]imidazole-4-carboxamide
isomerase (EC 5.3.1.16)
The reaction proceeds as according to:
phosphoribosylformiminoAICAR-phosphate => phosphoribulosylformimino-AICAR-P
No effectors have been identified for fungi.
r11.32: Imidazole glycerol phosphate synthase (EC 2.4.2.-)
Subsequently phosphoribulosylformimino-AICAR-P converts into:
phosphoribulosylformimino-AICAR-P + gln => glu + AICAR + D-erythro-imidazole-glycerol-phosphate
This enzyme is not in the databases of KEGG and Brenda.
r11.32: Imidazoleglycerol-phosphate dehydratase (EC 4.2.1.19)
This enzyme catalyzes the following reaction:
D-erythro-imidazole-glycerol-phosphate => imidazole acetol-phosphate + H2O
Table 18.42: Connectivity for imidazoleglycerol-phosphate dehydratase
effector
organism
comment
cys
Saccharomyces cerevisiae
activator
Pi
Saccharomyces cerevisiae
inhibitor; Ki = 0.008 mM
Other activating compounds are 2-Mercaptoethanol, mercaptoethylamine and D-erythro-imidazole-glycerolphosphate, whereas 3-Amino-1,2,4-triazole is known as an inhibitor. However none of these metabolites is included
in the stoichiometric model.
r11.32: Histidinol-phosphate transaminase (EC 2.6.1.9)
The following reversible reaction occurs by the catalysis of this enzyme:
imidazole acetol-phosphate + glu => L-histidinol phosphate + aKG
Table 18.43: Connectivity for histidinol-phosphate transaminase
effector
organism
comment
aKG
Neurospora sp.
activator for reverse reaction; Km = 1.2 mM
Moreover histidinol phosphate is also identified as an activator for the reserve reaction (Km = 1.1 mM) but this
metabolite is not included in the stoichiometric model.
r11.32: Histidinol-phosphatase (EC 3.1.3.15)
This enzyme catalyzes the following reaction:
L-histidinol phosphate + H2O = L-histidinol + Pi
A few inhibitors for baker’s yeast are included in the Brenda database such as arsenate, ethylenimine, iodoacetamide,
iodoacetate, N-ethylmaleimide and p-chloromercuribenzoate, L-Histidinol phosphate enhances the activity in baker’s
yeast (0.1<Km<0.25 mM) and in Neurospora crassa (Km = 4.2 mM). But since none of these compounds are
included in the stoichiometric model, their influence on the activity won’t be included in the model.
r11.32: histidinol dehydrogenase (EC 1.1.1.23)
- 63 -
The enzyme catalyzes the following reactions
L-histidinol + NAD => histidinal + NADH
L-histidinal + NAD + H2O => his + NADH
No effectors have been found for fungi.
The sum of the intermediate reactions is:
PRPP + ATP + 3 H2O + gln + 2 NAD => 2 PP + AICAR + aKG + Pi + his + 2 NADH
Although this is the only pathway in the Metacyc database for the synthesis of histidine, the above reaction is
unequal to r11.32. The main difference is that AICAR, which is also involved in r13.1, is not included in the
stoichiometric model which suggests that there exist a reaction in which AICAR can be lumped to metabolites which
is included in the model. Subtracting the above reaction from r11.32 yields:
2 ATP + CO2 + NADPH + NH4 + AICAR => 3 ADP + NADP + 2 H2O + Pi
This reaction has not yet been found in any of the databases.
r11.33: 0.137 ala + 0.0273 arg + 0.0358 asn + 0.061 asp + 0.00526 cys + 0.175 gln + 0.267 glu + 0.0336 gly +
0.0294 his + 0.0137 ile + 0.0168 leu + 0.0231 lys + 0.00526 met + 0.00526 phe + 0.0578 pro + 0.0494 ser + 0.0326
thr + 0.0021 trp + 0.00631 tyr + 0.0168 val => 4.56 AApool
No enzymes have been found in any of the databases which catalyze this reaction.
r11.34: 0.134 ala + 0.07 arg + 0.0386 asn + 0.0386 asp + 0.02 cys + 0.0509 gln + 0.0509 glu + 0.132 gly + 0.0119
his + 0.0307 ile + 0.0559 leu + 0.0348 lys + 0.0103 met + 0.0266 phe + 0.0537 pro + 0.044 ser + 0.0467 thr + 0.01
trp + 0.0203 tyr + 0.12 val => 4.53 ExPept + H2O
No enzymes have been found in any of the databases which catalyze this reaction.
19 Protein synthesis
12.1: 0.113 ala + 0.0572 arg + 0.038 asn + 0.038 asp + 0.00459 cys + 0.0503 gln + 0.0503 glu + 0.102 gly + 0.025
his + 0.0486 ile + 0.0784 leu + 0.0485 lys + 0.0133 met + 0.0478 phe + 0.0554 pro + 0.0561 ser + 0.0608 thr +
0.0099 trp + 0.0242 tyr + 0.0785 val => AAprotsyn
No enzymes have been found in any of the databases which catalyze this reaction.
r12.2: AAprotsyn + 4 ATP + 3 H2O => 4 ADP + 4 H + 4 Pi + 4.81 PROT
No enzymes have been found in any of the databases which catalyze this reaction.
20 Nucleotide biosynthesis
20.1
Purine metabolism (IMP synthesis)
r13.1: 1 asp + 4 ATP + CO2 + 2 FTHF + 2 gln + 1 gly + 2 H2O + PRPP => 4 ADP + fum + 2 glu + 8 H + IMP +
6 Pi + + 2 THF
AMP
inhibits
amidophosphoribosyltransferase,
adenylosuccinate lyase.
phosphoribosylformylglycinamidine
cyclo-ligase
and
Code:
r13.1a
Enzyme:
amidophosphoribosyltransferase
EC number:
2.4.2.14
Reaction Brenda: PRPP + H2O + gln => 5-phosphoribosylamine + glu + PP
Reversibility:
irreversible
Act & Inh:
Table 20.1
Eff. NAS:
GMP (Schizosaccharomyces pombe)
Turnover nr:
unknown
- 64 -
Spec activity:
Mechanism:
Location:
0.02 μmol/min/mg (Schizosaccharomyces pombe)
unknown
cytosol
Table 20.1: Connectivity for amidophosphoribosyltransferase
effector
organism
comment
AMP
Schizosaccharomyces p.
inhibitor
Code:
r13.1b
Enzyme:
phosphoribosylamine-glycine ligase
EC number:
6.3.4.13
Reaction Brenda: FTHF + 5-phosphoribosylamine => THF + GAR
Reversibility:
irreversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Code:
r13.1c
Enzyme:
phosphoribosylglycinamide formyltransferase
EC number:
2.1.2.2
Reaction Brenda: FTHF + GAR => THF + N2-formyl-N1-(5-phospho-D-ribosyl)glycinamide
Reversibility:
irreversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Code:
r13.1d
Enzyme:
phosphoribosylformylglycinamidine synthase
EC number:
6.3.5.3
Reaction Brenda: ATP + N2-formyl-N1-(5-phospho-D-ribosyl)glycinamide + gln + H2O => ADP + Pi
+ FGAM + glu
Reversibility:
irreversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Code:
r13.1e
Enzyme:
phosphoribosylformylglycinamidine cyclo-ligase
EC number:
6.3.3.1
Reaction Brenda: ATP + FGAM = ADP + Pi + AIR
Reversibility:
irreversible
Act & Inh:
Table 20.2
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
- 65 -
Table 20.2: Connectivity for phosphoribosylformylglycinamidine cyclo-ligase
effector
organism
comment
AMP
Saccharomyces cerevisiae
inhibitor, competitive with ATP
Code:
r13.1f
Enzyme:
phosphoribosylaminoimidazole carboxylase
EC number:
4.1.1.21
Reaction Brenda: AIR + CO2 => phosphoribosyl-carboxy-aminoimidazole
Reversibility:
reversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Code:
r13.1g
Enzyme:
phosphoribosylaminoimidazolesuccinocarboxamide synthase
EC number:
6.3.2.6
Reaction Brenda: ATP + phosphoribosyl-carboxy-aminoimidazole + asp = ADP + Pi + 5'phosphoribosyl-4-(N-succinocarboxamide)-5-aminoimidazole
Reversibility:
reversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Code:
r13.1h
Enzyme:
adenylosuccinate lyase
EC number:
4.3.2.2
Reaction Brenda: 5'-phosphoribosyl-4-(N-succinocarboxamide)-5-aminoimidazole => fum + AICAR
Reversibility:
reversible
Act & Inh:
Table 20.3
Eff. NAS:
ammonium
salt
of
N6-malonyl
adenosine
5'-phosphate,
N-(5-amino-1-(beta-Dribofuranosyl)imidazole-4-carbonyl)-L-threo-beta-methylaspartic acid 5'-phosphate
2',3'dideoxyadenylosuccinate,
N6-(1,2-dicarboxyethyl)AMP
and
virazole
5'-phosphate
(Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Table 20.3: Connectivity for adenylosuccinate lyase
effector
organism
comment
AMP
Saccharomyces cerevisiae
Km = 0.048 mM; reverse reaction
fum
Saccharomyces cerevisiae
Km = 0.52 mM; reverse reaction
Code:
r13.1i
Enzyme:
phosphoribosylaminoimidazolecarboxamide formyltransferase
EC number:
2.1.2.3
Reaction Brenda: AICAR + FTHF => THF + 5-formamido-1-(5-phospho-D-ribosyl)imidazole-4carboxamide
Reversibility:
irreversible
- 66 -
Act & Inh:
Eff. NAS:
Turnover nr:
Spec activity:
Mechanism:
Location:
none found
none found
unknown
unknown
unknown
cytosol
Code:
r13.1j
Enzyme:
IMP cyclohydrolase
EC number:
3.5.4.10
Reaction Brenda: 5-formamido-1-(5-phospho-D-ribosyl)imidazole-4-carboxamide => IMP + H2O
Reversibility:
reversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
The sum of all intermediate reactions is:
PRPP + H2O + 2 gln + 4 ATP + gly + 2 FTHF + CO2=> 2 glu + PP + 4 ADP + 4 Pi + 2 THF + fum
Eliminating pyrophosphate with reachtion l2 yields reaction r13.1
r13.2: asp + ATP + IMP => ADP + AMP + fum + 2 H + Pi
Code:
r13.2a
Enzyme:
adenylosuccinate lyase
EC number:
6.3.4.4
Reaction Brenda: GTP + IMP + asp => GDP + Pi + adenylosuccinate
Reversibility:
irreversible
Act & Inh:
Table 20.4
Eff. NAS:
5-amino-4-(N-succinocarboxamide)imidazole ribonucleotide, 5-amino-4-carbamoyl-imidazole
ribonucleotide, hydroxylamine, GTP, adenylosuccinate, oligonucleotides and aspartate analogs
(Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Table 20.4: Connectivity for adenylosuccinate lyase
effector
organism
comment
AMP
Saccharomyces cerevisiae
inhibitor ; Ki = 6 mM
Schizosaccharomyces p.
inhibitor
ATP
Saccharomyces cerevisiae
inhibitor; Ki = 4 mM
asp
Saccharomyces cerevisiae
1< Km< 6.7 mM
Schizosaccharomyces p.
activator; Km = 1.5 mM
IMP
Saccharomyces cerevisiae
0.2 < Km < 1.7 mM
Schizosaccharomyces p.
0.037 < Km < 0.07 mM
GMP
Saccharomyces cerevisiae
inhibitor
Schizosaccharomyces p.
inhibitor
The second step is catalyzed by adenylosuccinate lyase which already been discussed in r13.1h. However the
reaction is now catalyzing the following reaction
adenylosuccinate => fum + AMP
The sum of the intermediate reactions is:
- 67 -
GTP + IMP + asp => GDP + Pi + fum + AMP
r13.3: 2 ATP + gln + 3 H2O + IMP + NAD => 2 ADP + glu + GMP + 4H + NADH + 2 Pi
Code:
r13.3a
Enzyme:
IMP dehydrogenase
EC number:
1.1.1.205
Reaction Brenda: IMP + NAD + H2O = xanthosine 5'-phosphate + NADH + H
Reversibility:
irreversible
Act & Inh:
Table 20.5
Eff. NAS:
GDP, GTP (Schizosaccharomyces pombe)
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Table 20.5: Connectivity for IMP dehydrogenase
effector
organism
comment
ATP
Schizosaccharomyces p.
30% activation at 0.8 mM
CMP
Schizosaccharomyces p.
25% activation at 0.6 mM
GMP
Schizosaccharomyces p.
product inhibition; 55% inhibition at 0.6 mM
UMP
Schizosaccharomyces p.
25% inhibition at 0.6 mM
Code:
r13.3b
Enzyme:
GMP synthase (glutamine-hydrolysing)
EC number:
6.3.5.2
Reaction Brenda: ATP + xanthosine 5'-phosphate + gln + H2O => AMP + PP + GMP + glu
Reversibility:
irreversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown.
Location:
cytosol
The sum of the partial reactions is therefore:
IMP + gln +NAD + 2 H2O + ATP => NADH + H + AMP + PP + GMP + glu
Eliminating pyrophosphate with reaction l2 yields reaction r13.3.
r13.8: A + 2 ATP => 3 ADP + H
Code:
r13.8
Enzyme:
adenosine kinase
EC number:
2.7.1.20
Reaction Brenda: A + ATP => ADP + AMP
Reversibility:
reversible
Act & Inh:
Table 20.6
Eff. NAS:
cytidine, guanosine, inosine and uridine (Saccharomyces cerevisiae)
Turnover nr:
25.5 s-1 (Saccharomyces cerevisiae with A as substrate)
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Table 20.6: Connectivity for adenosine kinase
effector
organism
comment
A
Saccharomyces cerevisiae
0.003<Km<0.0035 mM
- 68 -
ATP
Saccharomyces cerevisiae
Km = 0.1 mM
Elimination of AMP with reaction l1 will yield reaction r13.8
20.2 Pyrimidine metabolism
r13.4: asp + 2 ATP + gln + 2 H2O + NAD + PRPP => 2 ADP + glu + 4 H + NADH + 4 Pi + UMP
Carbamoyl-phosphate synthase is inhibited by UTP and activated by IMP and phosphate. Rib5P and E4P inhibit
orotate phosphoribosyltransferase at high concentrations, but it will be assumed that these conditions do not apply in
a physiologically environment within the cell.
Code:
r13.4a
Enzyme:
carbamoyl-phosphate synthase (glutamine-hydrolysing)
EC number:
6.3.5.5
Reaction Brenda: 2 ATP + gln + CO2 + H2O => 2 ADP + Pi + glu + carbP
Reversibility:
irreversible
Act & Inh:
Table 20.7
Eff. NAS:
L-ornithine (Saccharomyces cerevisiae chimeric enzyme)
Turnover nr:
unknown
Spec activity:
509 μmol/min/mg
Mechanism:
unknown
Location:
cytosol
Table 20.7: Connectivity for carbamoyl-phosphate synthase
effector
organism
comment
UMP
Saccharomyces cerevisiae
inhibitor
UTP
Saccharomyces cerevisiae
inhibitor
IMP
Saccharomyces cerevisiae
activator
Pi
Saccharomyces cerevisiae
activator
Code:
r13.4b
Enzyme:
aspartate carbamoyltransferase
EC number:
2.1.3.2
Reaction Brenda: carbP + asp = Pi + carbamoyl-asp
Reversibility:
irreversible
Act & Inh:
Table 20.8
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Table 20.8: Connectivity for aspartate carbamoyltransferase
effector
organism
comment
UTP
Saccharomyces cerevisiae
inhibitor
Code:
r13.4c
Enzyme:
dihydroorotase
EC number:
3.5.2.3
Reaction Brenda: carbamoyl-asp => (S)-dihydroorotate + H2O
Reversibility:
reversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
- 69 -
Spec activity:
Mechanism:
Location:
unknown
unknown
cytosol
Subsequently orotate will be synthesized. Orotate reductase (EC 1.3.1.14) catalyzes the following reaction:
(S)-dihydroorotate + NAD = orotate + NADH + H
No effectors have been identified.
Code:
r13.4d
Enzyme:
orotate reductase (NADH)
EC number:
1.3.1.14
Reaction Brenda: (S)-dihydroorotate + NAD => orotate + NADH +H
Reversibility:
reversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Code:
r13.4e
Enzyme:
orotate phosphoribosyltransferase
EC number:
2.4.2.10
Reaction Brenda: orotate + PRPP=> orotidine-5P + PP
Reversibility:
reversible
Act & Inh:
Table 20.9
Eff. NAS:
anthranilate, arabinose-5P, f1P, nicotinate and orotidylate, orotate, PP and orotidine-5P
(Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
17-81.6 μmol/min/mg
Mechanism:
unknown
Location:
unknown
Table 20.9: Connectivity for orotate phosphoribosyltransferase
effector
organism
comment
PRPP
Saccharomyces cerevisiae
product inhibition in reverse reaction; 0.038<Km<0.062
mM;
Pi
Saccharomyces cerevisiae
inhibition at high concentration
Rib5P
Saccharomyces cerevisiae
inhibition at high concentration
E4P
Saccharomyces cerevisiae
inhibition at high concentration
Code:
r13.4f
Enzyme:
orotidine-5'-phosphate decarboxylase
EC number:
4.1.1.23
Reaction Brenda: orotidine-5P => UMP + CO2
Reversibility:
irreversible
Act & Inh:
Table 20.10
Eff. NAS:
1-(5'-Phospho-beta-D-ribofuranosyl)barbituric acid, 1-ribosyloxipurinol 5'-phosphate, uridine5’phosphate derivates, 5,5'-dithiobis(2-nitrobenzoate), PCMB, xanthosine 5’-phosphate and
orotidine-5P (Saccharomyces cerevisiae)
Turnover nr:
44 s-1 (Saccharomyces cerevisiae)
Spec activity:
27.2-90 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
Table 20.10: Connectivity for orotidine-5'-phosphate decarboxylase
- 70 -
effector
Pi
Rib5P
UMP
organism
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
comment
weak inhibitor
competitive inhibitor ; 0.08<Ki<0.5 mM
competitive inhibitor ; 0.092<Km<0.37 mM
The overall reaction therefore becomes:
2 ATP + gln + asp + NAD + PRPP => 2 ADP + 2 Pi + glu + NADH + H + PP + UMP
This reaction is not entirely the same as r13.4 in which 2 water molecules are consumed. Hydrolyzing PP into two
phosphates only account for one water molecule. It is unknown in which reaction the other water molecule is
involved.
r13.5: 2 ATP + UMP => 2 ADP + UTP
Code:
r13.5a
Enzyme:
cytidylate kinase
EC number:
2.7.4.14
Reaction Brenda: ATP + UMP => ADP + UDP
Reversibility:
reversible
Act & Inh:
Table 20.11
Eff. NAS:
NEM, dithiothreitol and PCMB (Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
11.1 mmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
Other acceptors for cytidylate kinase are CMP and dCMP.
Table 20.11: Connectivity for cytidylate kinase
effector
organism
comment
CMP
Saccharomyces cerevisiae
competitive inhibition with UMP; Km = 0.071 mM
UMP
Saccharomyces cerevisiae
Km = 0.052 mM
Code:
r13.5b
Enzyme:
nucleoside-diphosphate kinase
EC number:
2.7.4.6
Reaction Brenda: ATP + UDP => ADP + UTP
Reversibility:
reversible
Act & Inh:
Table 20.12
Eff. NAS:
(d)CDP, dGDP and dTDP (Saccharomyces cerevisiae)
Turnover nr:
13.1 (Saccharomyces cerevisiae on dTDP as a substrate)
Spec activity:
5.4 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
unknown
Table 20.12: Connectivity for nucleoside-diphosphate kinase
effector
organism
comment
(d)UDP
Saccharomyces cerevisiae
Km = 0.28 mM
The overall reaction is:
2 ATP + UMP => 2 ADP + UTP
r13.6: ATP + H2O + gln + UTP > ADP + Pi + CTP + 2 H + glu
Code:
Enzyme:
r13.6
CTP synthase
- 71 -
EC number:
6.3.4.2
Reaction Brenda: ATP + UTP + gln + H2O => ADP + Pi + CTP + glu
Reversibility:
reversible [4], irreversible [2]
Act & Inh:
Table 20.13
Eff. NAS:
NEM, p-chloromercuribenzenesulfonic acid, 2-mercaptoethanol [1], GTP [4] (Saccharomyces
cerevisiae)
Turnover nr:
unknown
Spec activity:
0.2-2.5 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
unknown
Although glutamine is employed as substrate in the stoichiometric model, CTP synthase also can utilize ammonia as
substrate [2]. The hill coefficient of CTP synthase is 1.4.
Table 20.13: Connectivity for CTP synthase
effector
organism
comment
CTP
Saccharomyces cerevisiae
allosteric inhibition [1]; IC = 0.3 mM [4]
UTP
Saccharomyces cerevisiae
0.04<Km<0.1mM [1]; 0.07<Km<0.18 mM [4]
ATP
Saccharomyces cerevisiae
0.44<Km<0.57 mM [4]
r13.7: 2 ADP + CTP => ADP + CMP
This reaction is catalyzed by cytidylate kinase (EC 2.7.4.14) and nucleoside-diphosphate kinase (EC 2.7.4.6) which
also have been employed for reaction r13.5. However these enzymes now catalyse respectively:
ATP + CMP => ADP + CDP
ATP + CDP => ADP + CTP
Summing these two partial reactions will yield reaction r13.7 but in opposite reaction.
r13.9: ATP + UDP => ADP + UTP
Nucleoside-diphosphate kinase (EC 2.7.4.6) has already been discussed for reaction r13.5 & r13.7. Reaction r13.9 is
the same as r13.5b.
21 RNA synthesis
r14.1: 0.349 AMP + 3.23 ATP + 0.168 CMP + 0.26 GMP + 2.23 H2O + 0.222 UMP => 3.23 ADP + 3.23 H +
3.23 Pi + 9.61 RNA
No enzymes have been found in any of the databases which catalyze this reaction and as a consequence no effectors
have been identified.
22 ATP hydrolysis
r15.1: ATP + H2O => ADP + H + Pi
Code:
r15.1a
Enzyme:
non-chaperonin molecular chaperone ATPase
EC number:
3.6.4.10
Reaction Brenda: ATP + H2O => ADP + H + Pi
Reversibility:
irreverisible
Act & Inh:
none found
Eff. NAS:
17-allyl-amino-17-demethoxygeldanamycin, geldanamycin, guanidinium chloride, radicicol, 15deoxyspergualin, activator of Hsp90 ATPase, DnaJ and Sti1 (Saccharomyces cerevisiae)
Turnover nr:
0.0078-1.2 μmol/min/mg (Saccharomyces cerevisiae)
Spec activity:
unknown
- 72 -
Mechanism:
Location:
unknown
cytosol
23 Synthesis of fatty acids
23.1
Inositol phosphate metabolism
r16.1: g6P + H2O => ino + Pi
Code:
r16.1a
Enzyme:
inositol-3-phosphate synthase
EC number:
5.5.1.4
Reaction Brenda: g6P => 1D-myo-inositol 3-phosphate
Reversibility:
irreversible
Act & Inh:
Table 23.1
Eff. NAS:
1-deoxy-1-(phosphonomethyl)myo-2-inosose, 2-deoxy-D-glucitol (Ki = 0.00067 mM), 2-deoxy-Dglucose 6-phosphate, 2-deoxy-myo-inositol 1-phosphate, dihydroxyacetone phosphate and myo-2Inosose 1-phosphate (Saccharomyces cerevisiae), EDTA (Neurospora crassa)
Turnover nr:
unknown
Spec activity:
0.086-0.22 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
Table 23.1: Connectivity for inositol-3-phosphate synthase
effector
organism
comment
NH4
Saccharomyces cerevisiae
strong activator
Neurospora crassa
activator
g6P
Saccharomyces cerevisiae
1.18<Km<1.9 mM
Candida utilis
Km = 0.06 mM
PP
Neurospora crassa
inhibitor in the presence of NH4
Code:
r16.1b
Enzyme:
Inositol-1(or 4)-monophosphatase
EC number:
3.1.3.25
Reaction Brenda: myo-inositol 3-phosphate + H2O = ino + Pi
Reversibility:
irreversible
Act & Inh:
Table 23.2
Eff. NAS:
inositol-3P and 2-deoxyglucose-6-phosphate (Candida utilis)
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Inositol-1(or 4)-monophosphatase will act on all six
except
myo-inositol
2-phosphate,
but
will
containing more than one phosphate group as substrate.
isomers
not
of
myo-inositol
accept
a
phosphate, all
myo-inositol
Table 23.2: Connectivity for inositol-1(or 4)-monophosphatase
effector
organism
comment
Rib5P
Candida utilis
inhibitor
gly
Saccharomyces cerevisiae
depresses enzyme expression if present in culture
glu
Saccharomyces cerevisiae
depresses enzyme expression if present in culture
6Pgluct
Candida utilis
slight inhibitor
- 73 -
The overall reaction becomes therefore:
g6P + H2O => ino + Pi
23.2
Glycerophospolipid metabolism
r16.2: GAP + H + NADH => gcl3P + NAD
Code:
r16.2a
Enzyme:
triose-phosphate isomerase
EC number:
5.3.1.1
Reaction Brenda: reversible
Reversibility:
GAP => glycerone-phosphate
Act & Inh:
Table 23.3
Eff. NAS:
2,4-Dinitrofluorobenzene,
5,5'-dithiobis(2-nitrobenzoate),
acetylphosphate,
D-alphaglycerophosphate, iodoacetate, methyl methanethiosulfonate, S-Phenyl-p-toluenethiosulfonate and
phosphoglycolohydroxamate, glycerone-phosphate (Saccharomyces cerevisiae)
Turnover nr:
16700 s-1 (Saccharomyces cerevisiae)
Spec activity:
10000-19250 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
Table 23.3: Connectivity for triose-phosphate isomerase
effector
organism
comment
GAP
Saccharomyces cerevisiae
Km = 1.27 mM
Pi
Saccharomyces cerevisiae
competitive inhibitor
PEP
Saccharomyces cerevisiae
competitive inhibitor
Code:
r16.2b
Enzyme:
glycerol-3-phosphate dehydrogenase (NAD+)
EC number:
1.1.1.18
Reaction Brenda: glycerone phosphate + NADH + H => gcl3P + NAD
Reversibility:
reversible
Act & Inh:
Table 23.4
Eff. NAS:
TEA-buffer, glycerone phosphate (Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
41.1-2396 mmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
equilibrium random-bi-bi reaction mechanism (Saccharomyces cerevisiae)
Location:
cytosol
Table 23.4: Connectivity for glycerol-3-phosphate dehydrogenase
effector
organism
comment
ATP
Saccharomyces cerevisiae
90% inhibition at physiological concentration, i.e. 10
mM
ADP
Saccharomyces cerevisiae
90% inhibition at physiological concentration, i.e. 10
mM
f16P
Saccharomyces cerevisiae
non-competitive inhibitor at physiological concentrations
NAD
Saccharomyces cerevisiae
competitive inhibitor to NADH at physiological
concentration
Pi
Saccharomyces cerevisiae
competitive inhibitor against glycerone phosphate, noncompetitive inhibitor against NADH
glc3P
Saccharomyces cerevisiae
Km = 34 mM
NADH
Saccharomyces cerevisiae
Km = 0.024 mM
r16.6: gcl3P + linCoA + olCoA => 2 HCoA + phospht
- 74 -
Code:
r16.6a
Enzyme:
glycerol-3-phosphate O-acyltransferase
EC number:
2.3.1.15
Reaction Brenda: (1) acyl-CoA + glc3P = HCoA + 1-acyl-glc3P
(2) linCoA + glc3P => HCoA + 1-linoleoyl-glc3P
(3) olCoA + glc3P => HCoA + 1-oleoyl-glc3P
Reversibility:
reversible
Act & Inh:
Table 23.5
Eff. NAS:
N-ethylmaleimide, palmitoyl-CoA, trypsin,
(Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
glycerone
phosphate
and
deoxycholate
This enzyme only accepts derivatives of fatty acids of a chain length above C10 [2] such as oleoyl and linoleoyl
derivates. A few compounds are mentioned in Brenda database that influence the activity. For baker yeast they are
deoxycholate, palmitoyl-CoA, trypsin, glycerone phosphate (Ki=0.54 mM) and Triton X-100. However none of
these components are included in the stoichiometric model.
Table 23.5: Connectivity for glycerol-3-phosphate O-acyltransferase
effector
organism
comment
gcl3P
Saccharomyces cerevisiae
Km = 0.03 mM
Code:
r16.6b
Enzyme:
1-acylglycerol-3-phosphate O-acyltransferase
EC number:
2.3.1.51
Reaction Brenda: (1) acyl-CoA + 1-acyl-sn-glycerol 3-phosphate => HCoA + 1,2-diacyl-glc3P
(2) linoleoyl-CoA + 1-oleoyl-sn-glycerol 3-phosphate => HCoA + 1-oleoyl-2-linoleoyl-glc3P
Reversibility:
irreversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Phosphatidate is a synonym for any of the 1,2-diacyl-sn-glycerol 3-phosphates. Saturated and unsaturated acyl-CoAs
are converted at comparable rates by this enzyme. Moreover it has a very broad specificity for acyl-CoAs.
The overall reaction will therefore become:
glc3P + olCoA + linCoA => 2HCoA + phospht
r16.7: CTP + H2O + phospht => 2 Pi + CDPDAcglc
The non-zero elasticities with respect to the reactants are εr16.7,CTP:cyt, εr16.7,phospht:cyt, εr16.7,Pi:cyt and εr16.7,CDPDAglc:cyt.
Code:
r16.7
Enzyme:
phosphatidate cytidylyltransferase
EC number:
2.7.7.41
Reaction Brenda: CTP + phospht => PP + CDPDAcglc
Reversibility:
reversible (Saccharomyces cerevisiae)
Act & Inh:
Table 23.6
Eff. NAS:
dCTP, PCMB, triton and thiophosphatidate
Turnover nr:
8.92 s-1 (Saccharomyces cerevisiae)
Spec activity:
0.45-1.409 μmol/min/mg (Saccharomyces cerevisiae)
- 75 -
Mechanism:
Location:
unknown
cytosol
Table 23.6: Connectivity for phosphatidate cytidylyltransferase
effector
organism
comment
CTP
Saccharomyces cerevisiae
Km = 1 mM
phospht
Saccharomyces cerevisiae
Km = 0.5 mM
Pi
Saccharomyces cerevisiae
for PP, product inhibition
r16.8: CDPDAcglc + ser => CMP + PHser
Code:
r16.8
Enzyme:
CDP-diacylglycerol-serine O-phosphatidyltransferase
EC number:
2.7.8.8
Reaction Brenda: CDPDAcglc + ser => CMP + PHser
Reversibility:
irreversible
Act & Inh:
Table 23.7
Eff. NAS:
cardiolipin, p-hydroxymercuribenzoate, diacylglycerol,
(Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
2.3 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
bi-bi sequential mechanism
Location:
cytosol
EDTA
and
2-mercaptoethanol
Table 23.7: Connectivity for CDP-diacylglycerol-serine O-phosphatidyltransferase
effector
organism
comment
ser
Saccharomyces cerevisiae
0.058<Km<13 mM
CDPDAcglc Saccharomyces cerevisiae
0.06<Km<0.12 mM
ino
Saccharomyces cerevisiae
non-competitive inhibitor; Ki = 0.065 mM
PHino
Saccharomyces cerevisiae
activator
PHchol
Saccharomyces cerevisiae
activator
phospht
Saccharomyces cerevisiae
activator
r16.9: H + PHser => PHeta + CO2
Code:
r16.9
Enzyme:
phosphatidylserine decarboxylase
EC number:
4.1.1.65
Reaction Brenda: PHser => PHeta + CO2
Reversibility:
irreversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
r16.10: PHeta + 3 SAM => 3 H + PHchol + 3 SAH
Code:
r16.10
Enzyme:
phosphatidylethanolamine N-methyltransferase
EC number:
2.1.7.17
Reaction Brenda: (1) PHeta + SAM => SAH + phosphatidyl-N-methylethanolamine
(2) phosphatidyl-N-methylethanolamine + SAM => SAH + phosphatidyl-Ndimethylethanolamine
- 76 -
Reversibility:
Act & Inh:
Eff. NAS:
Turnover nr:
Spec activity:
Mechanism:
Location:
(3) phosphatidyl-N-dimethylethanolamine + SAM => SAH + PHchol
irreversible
Table 23.8
PCMB (Saccharomyces cerevisiae)
unknown
unknown
unknow
cytosol
Table 23.8: Connectivity for phosphatidyl-N-methylethanolamine N-methyltransferase
effector
organism
comment
SAH
Saccharomyces cerevisiae
product inhibition, 0.012<Km<0.17 mM
SAM
Saccharomyces cerevisiae
Km = 0.11 mM
PHeta
Saccharomyces cerevisiae
Km = 0.057 mM
The overall reaction is:
PHeta + 3 SAM => PHchol + 3 SAH
r16.11: CPDDAcgcl + ino => CMP + H + PHino
Code:
r16.11
Enzyme:
CDP-diacylglycerol-inositol 3-phosphatidyltransferase
EC number:
2.7.8.11
Reaction Brenda: CDPDAcglc + ino => PHino + CMP
Reversibility:
irreversible
Act & Inh:
Table 23.9
Eff. NAS:
CDP, CTP, GTP, triton X-100, TTP and 2-deoxy-CDP-diacylglycerol (Candida albicans), pchloromercuribenzene sulfonate (Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
0.001-0.002 μmol/min/mg (Candida albicans), 0.0008-0.08 μmol/min/mg (Saccharomyces
cerevisiae)
Mechanism:
unknown
Location:
cytosol
Table 23.9: Connectivity for CDP-diacylglycerol-inositol 3-phosphatidyltransferase
effector
organism
comment
CDPDAcglc Saccharomyces cerevisiae
Km = 0.07 mM
Candida albicans
Km = 0.036 mM
CMP
Saccharomyces cerevisiae
reverse reaction, Km = 0.022 mM
ino
Saccharomyces cerevisiae
0.066<Km<0.277 mM
Candida albicans
Km = 0.55 mM
ADP
Candida albicans
inhibitor
ATP
Candida albicans
inhibitor
Pi
Candida albicans
inhibitor; PP is efficient inhibitor
UDP
Candida albicans
inhibitor
UTP
Candida albicans
inhibitor
23.3
Glycerolipid metabolism
r16.12: H2O + phospht + steaCoA => HCoA + Pi + TRIA
Code:
r16.12a
Enzyme:
phosphatidate phosphatase
EC number:
3.1.3.4
Reaction Brenda: phospht + H2O = 1,2-diacylglycerol + Pi
- 77 -
Reversibility:
Act & Inh:
Eff. NAS:
Turnover nr:
Spec activity:
Mechanism:
Location:
irreversible
Table 23.10
diacylglycerol diphosphate, NEM, p-chloromercuriphenylsulfonic acid, phenylglyoxal,
phosphatidic acid,
propanolol, psychosine, sphinganine and sphingosine, triton X-100
(Saccharomyces cerevisiae)
unknown
1.37-2.35 μmol/min/mg (Saccharomyces cerevisiae)
unknown
cytosol
Table 23.10: Connectivity for phosphatidate phosphatase
effector
organism
comment
phospht
Saccharomyces cerevisiae
Km = 0.05 mM
Code:
r16.12b
Enzyme:
diacylglycerol O-acyltransferase
EC number:
2.3.1.20
Reaction Brenda: steaCoA + 1,2-diacylglycerol = HCoA + TRIA
Reversibility:
irreversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
0.0018 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
Only long chain acyl-CoAs, such as steaCoA, are accepted by diacylglycerol O-acyltransferase.
23.4
Miscellanous
r16.3: 9 AcCoA + 8 ATP + 8 H + 16 NADPH => 8 ADP + 8 HCoA + 16 NADP + 8 Pi + steaCoA
This reaction hasn’t been found in the online databases.
r16.4: H + NADH + O2 + steaCoA => 2 H2O + NAD + olCoA
No effectors have been found.
Code:
r16.4
Enzyme:
stearoyl-CoA 9-desaturase
EC number:
1.14.19.1
Reaction Brenda: steaCoA + NADH + H + O2 => olCoA + NAD + 2 H2O
Reversibility:
unknown
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
r16.5: H + NADH + O2 + olCoA => 2 H2O + linCoA + NAD
This reaction is not found explicitly in any of online databases. However stearoyl-CoA 9-desaturase, discussed in
r16.4, can react with acyl-CoA, acyl-acyl-carrier and acyl chains of phospholipids and may therefore catalyze this
reaction.
- 78 -
23.5
Biosynthesis of steriods
r16.13: 3 AcCoA + H + H2O + 2 NADPH => 3 HCoA + meva + 2 NADP
Code:
r16.13a
Enzyme:
acetyl-CoA C-acetyltransferase
EC number:
2.3.1.9
Reaction Brenda: 2 AcCoA => HCoA + acetoacetyl-CoA
Reversibility:
reversible
Act & Inh:
Table 23.11
Eff. NAS:
acetoacatyl-CoA (Candida tropicalis)
Turnover nr:
unknown
Spec activity:
29-59.8 μmol/min/mg (Candida tropicalis)
Mechanism:
unknown
Location:
cytosol
Table 23.11: Connectivity for acetyl-CoA C-acetyltransferase
effector
organism
comment
HCoA
Candida tropicalis
0.03< Km <0.05 mM
AcCoA
Candida tropicalis
0.69< Km <1.05 mM
Code:
r16.13b
Enzyme:
hydroxymethylglutaryl-CoA synthase
EC number:
2.3.3.10
Reaction Brenda: AcCoA + H2O + acetoacetyl-CoA => (S)-3-hydroxy-3-methylglutaryl-CoA + HCoA
Reversibility:
irreversible
Act & Inh:
Table 23.12
Eff. NAS:
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, acetyl-CoA analogues can act as substrates,
acetoacyl-analogues are no substrates, iodoacetamide, N-ethylmaleimide, p-chloromercuribenzoate
and Acetoacetyl-CoA (Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
2-2.1 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
Table 23.12: Connectivity for hydroxymethylglutaryl-CoA synthase
effector
organism
comment
HCoA
Saccharomyces cerevisiae
product inhibitor; 0.038<Km<0.06 mM
Pi
Saccharomyces cerevisiae
activator; potassium and sodium phosphate
SO4
Saccharomyces cerevisiae
activator
AcCoA
Saccharomyces cerevisiae
0.014<Km<0.018 mM
Code:
r16.13c
Enzyme:
hydroxymethylglutaryl-CoA reductase (NADPH)
EC number:
1.1.1.34
Reaction Brenda: (S)-3-hydroxy-3-methylglutaryl-CoA + 2 NADPH + 2 H => meva + HCoA +
2 NADP
Reversibility:
irreversible
Act & Inh:
unknown
Eff. NAS:
3-hydroxy-3-methylglutaryl-CoA, CoA disulfide, CoA disulfide, DTNB, glutathione and phydroxymercuribenzoate (Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
0.0035 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
- 79 -
The overall reaction is:
3 AcCoA + H2O + 2 NADPH=> 3 HCoA + meva + 2 NADP
r16.14: 18 ATP + 5 H2O + 6 meva + 2 NADPH + O2 => 18 ADP + 6 CO2 + 10 H + lano + 2 NADP + 18 Pi
Code:
r16.14a
Enzyme:
mevalonate kinase
EC number:
2.7.1.36
Reaction Brenda: ATP + meva = ADP + meva-5P
Reversibility:
irreversible
Act & Inh:
Table 23.13
Eff. NAS:
farnesyl diphosphate, geranyl diphosphate, geranylgeranyl diphosphate, p-chloromercuribenzoate,
phytyl diphosphate (Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
0.77 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
Table 23.13: Connectivity for mevalonate kinase
effector
organism
comment
UTP
Saccharomyces cerevisiae
activator, stimulation of ATP utilization
ATP
Saccharomyces cerevisiae
Km = 7.4 mM
Code:
r16.14b
Enzyme:
phosphomevalonate kinase
EC number:
2.7.4.2
Reaction Brenda: ATP + meva-5P =. ADP + (R)-5-diphosphomevalonate
Reversibility:
irreversible
Act & Inh:
Table 23.14
Eff. NAS:
none found
Turnover nr:
none found
Spec activity:
0.06 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
Table 23.14: Connectivity for phosphomevalonate kinase
effector
organism
comment
ATP
Saccharomyces cerevisiae
inhibitor above 10 mM
Code:
r16.14c
Enzyme:
diphosphomevalonate decarboxylase
EC number:
4.1.1.33
Reaction Brenda: ATP + (R)-5-diphosphomevalonate => ADP + Pi + isopentenyl diphosphate + CO2
Reversibility:
irreversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Code:
r16.14d
Enzyme:
isopentenyl-diphosphate DELTA-isomerase
EC number:
5.3.3.2
Reaction Brenda: isopentenyl diphosphate => dimethylallyl diphosphate
- 80 -
Reversibility:
Act & Inh:
Eff. NAS:
Turnover nr:
Spec activity:
Mechanism:
Location:
reversible
none found
isopentenyl diphosphate, 2-(dimethylamino)ethyl phosphate, 3,4-epoxy-1-butenyl diphosphate, 3(Fluoromethyl)-3-buten-1-yl diphosphate, iodoacetamide, isoamyl diphosphate and PCMB
(Saccharomyces cerevisiae)
unknown
0.109 μmol/min/mg (Saccharomyces cerevisiae)
unknown
cytosol
Subsequently the following irreversible reaction is catalyzed:
dimethylallyl diphosphate + isopentenyl diphosphate => PP + geranylgeranyl diphosphate
This reaction is catalyzed due to the involvement of farnesyltranstransferase (EC 2.5.1.29), geranyltranstransferase
(EC 2.5.1.10) and dimethylallyltranstransferase (EC 2.5.1.1).
Subsequently these enzymes are involved in the catalysis of the following reaction:
geranylgeranyl diphosphate + isopentenyl diphosphate => PP + E,E-farnesyl diphosphate
Code:
r16.14e
Enzyme:
geranyltranstransferase
EC number:
2.5.1.10
Reaction Brenda: (1) dimethylallyl diphosphate + isopentenyl diphosphate => PP + geranylgeranyl
diphosphate
(2) geranylgeranyl diphosphate + isopentenyl diphosphate => PP + E,E-farnesyl
diphosphate
Reversibility:
irreversible
Act & Inh:
none found
Eff. NAS:
dimethylallyl diphosphate and isopentenyl diphosphate (Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
2.33 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
Code:
r16.14f
Enzyme:
squalene synthase
EC number:
2.5.1.21
Reaction Brenda: (1) 2 farnesyl diphosphate => PP + presqualene diphosphate
(2) presqualene diphosphate + NADPH + H => squalene + PP + NADP
Reversibility:
irreversible
Act & Inh:
Table 23.15
Eff. NAS:
farnesyl diphosphate, ammonium analogues, deoxycholate, N-Ethylmaleimide
Turnover nr:
0.53 s-1 (Saccharomyces cerevisiae)
Spec activity:
0.95 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
NADH can also be employed as a cofactor.
Table 23.15: Connectivity for squalene synthase
effector
organism
comment
NADPH
Saccharomyces cerevisiae
0.004<Km<0.5 mM
NADH
Saccharomyces cerevisiae
0.004<Km<3.6 mM
NADP
Saccharomyces cerevisiae
inhibitor
Code:
Enzyme:
EC number:
r16.14g
squalene monooxygenase
1.14.99.7
- 81 -
Reaction Brenda: squalene + NADPH + H + O2 = (S)-squalene-2,3-epoxide + NADP + H2O
Reversibility:
irreversible
Act & Inh:
Table 23.16
Eff. NAS:
H2O2, terbinafine and triton X-100 and squalene (Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
3.5 * 10-5 - 0.0001 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
Table 23.16: Connectivity for squalene monooxygenase
effector
organism
Comment
O2
Saccharomyces cerevisiae
Km=0.0043 mM
Code:
r16.14h
Enzyme:
lanosterol synthase
EC number:
5.4.99.7
Reaction Brenda: (S)-squalene-2,3-epoxide = lano
Reversibility:
irreversible
Act & Inh:
none found
Eff. NAS:
many squalene derivates have been identified as effectors for baker’s yeast such as 2-aza-2,3dihydrosqualene, 19-azasqualene, 18-heptanor-2,3-oxidosqualene. However none of these
compounds are included in the stoichiometric model. The substrate (S)-squalene-2,3-epoxide is
also influencing the activity (Km = 0.035mM).
Turnover nr:
unknown
Spec activity:
0.0014 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Location:
cytosol
The overall reaction is:
2 NADPH + 2 H + O2 + 18 ATP + 6 meva => lano + 2 NADP + H2O + 6 PP + 6 Pi + 6 CO2 + 18 ADP
Eliminating pyrophosphate with reaction l2 will yields reaction r16.14
r16.15: lano + NAD + 2 THF => ergo + H + 2 MYTHF + NADH
No pathway in the Metacyc and KEGG database have been identified which corresponds to the given reaction in the
stoichiometric model. The pathway of the ergosterol synthesis in the metacyc database consists of the following
intermediate reactions:
lano + O2 + NADPH + H => 4,4-dimethyl-5-α-cholesta-8,14,24-trien-3-β-ol + NADP + formate
This reaction is catalyzed by sterol 14-demethylase (EC 1.14.13.70). Subsequently DELTA14-sterol reductase (EC
1.3.1.70) catalyzes the following reaction:
4,4-dimethyl-5-α-cholesta-8,14,24-trien-3-β-ol + NADPH => 4,4-dimethyl-5α-cholesta-8,24-dien-3-β-ol + NADP
C4 sterol methyl oxidase, no EC number assigned, catalyzes the following reaction:
4,4-dimethyl-5α-cholesta-8,24-dien-3-β-ol => 4-α-methyl zymosterol
Subsequently a spontaneous reaction occurs:
SAM + zymosterol => SAH + fecosterol
Fecosterol is degraded to episterol which is catalyzed by C8 sterol isomerase after which episterol is degradated into
ergostatrienol that is catalyzed by C5 sterol desaturase.
fecosterol => episterol
episterol + O2 + NADPH => 5,7,24(28)-ergostatrienol + 2 H2O + NADP
C22 sterol desaturase catalyzes the following reaction:
5,7,24(28)-ergostatrienol + O2 + NADPH => 5,7,22,24(28)-ergostatetraenol + 2 H2O + NADP
The final intermediate reaction is:
5,7,22,24(28)-ergostatetraenol + NADPH + H => ergosterol + NADP
Summing the intermediate reactions yields
lano + 3 O2 + 5 NADPH + 2 H + SAM => 5 NADP + formate + SAH + 4 H2O + ergo
- 82 -
This reaction is dissimilar to the given reaction in the stoichiometric model. No alternative pathways for the
biosynthesis of ergosterol have been found.
r16.16: ergo + olCoA => ESE + HCoA
This reaction hasn’t been found in the any of the online databases.
24 Synthesis of glycogen and polysaccharides
24.1
Aminosugar metabolism
r17.2: AcCoA + f6P + gln => chit + glu + H + HCoA + Pi
The above reaction consists of several lumped reactions. The pathway presented below for the biosynthesis of chitine
is deducted from the KEGG database for Saccharomyces cerevisiae. Note that chitine is a polymer that consists of
acetyl-D-glucosamine monomers.
Code:
r17.2a
Enzyme:
glutamine-fructose-6-phosphate transaminase (isomerizing)
EC number:
2.6.1.16
Reaction Brenda: gln + f6p = glu + D-glucosamine 6-P
Reversibility:
reversible
Act & Inh:
Table 24.1
Eff. NAS:
6-diazo-5-oxo-L-norleucine, azaserine (Neurospora crassa), L-2,3 diaminopropanoic acid, acetylL-2,3-diaminopropanoic acid derivates (Candida albicans), mercuric chloride, N-ethylmaleimide,
p-chloromercuribenzoate, UDP-N-acetylglucosamine and dithiothreitol (Saccharomyces
cerevisiae).
Turnover nr:
unknown
Spec activity:
0.00783 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
unknown
Compartment: cytosol
Table 24.1: Connectivity for glutamine-fructose-6-phosphate transaminase
effector
organism
Comment
f6P
Neurospora crassa
Km=0.75 mM
Code:
r17.2b
Enzyme:
glucosamine 6-phosphate N-acetyltransferase
EC number:
2.3.1.4
Reaction Brenda: AcCoA + D-glucosamine 6-P => HCoA + N-acetyl-D-glucosamine 6-P
Reversibility:
irreversible
Act & Inh:
Table 24.2
Eff. NAS:
p-chloromercuribenzoate, D-glucosamine 6-P and EDTA (Neurospora crassa).
Turnover nr:
unknown
Spec activity:
11.7-12.21 μmol/min/mg (Neurospora crassa)
Mechanism:
unknown
Compartment: cytosol
Table 24.2: Connectivity for glucosamine 6-phosphate N-acetyltransferase
effector
organism
Comment
AcCoA
Neurospora crassa
0.5<Km=0.78 mM
Code:
r17.2c
- 83 -
Enzyme:
phosphoacetylglucosamine mutase
EC number:
5.4.2.3
Reaction Brenda: N-acetyl-D-glucosamine 6-P => N-acetyl-α-D-glucosamine 1-P
Reversibility:
reversible
Act & Inh:
none listed in the Brenda database
Eff. NAS:
diethyldithiocarbamide (Neurospora crassa)
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Compartment: cytosol
Code:
Enzyme:
EC number:
Reaction Brenda:
Reversibility:
Act & Inh:
Eff. NAS:
Turnover nr:
Spec activity:
Mechanism:
Compartment:
r17.2d
UDP-N-acetylglucosamine diphosphorylase
2.7.7.23
UTP + N-acetyl-α-D-glucosamine 1-P => PP + UDP-N-acetyl-D-glucosamine
reversible
Table 24.3
5-hydroxyuridine, N-ethylmaleimide, p-chloromercuribenzoate, uridine, pseudo-uridine,
dithioerythritol, N-acetyl-D-glucosamine 1-P (0.011<Km<0.124 mM) and UDP-N-acetyl-Dglucosamine (Km=6.1 mM) (Saccharomyces cerevisiae).
15.4 s-1; wild type enzyme in Saccharomyces cerevisiae and N-acetyl-α-D-glucosamine 1-P as
substrate.
15.1 μmol/min/mg (Saccharomyces cerevisiae), 8.66 μmol/min/mg (Neurospora crassa)
unknown
cytosol
Table 24.3: Connectivity for UDP-N-acetylglucosamine diphosphorylase
effector
organism
Comment
Pi
Saccharomyces cerevisiae
product inhibition by PP; Km = 5 mM
Neurospora crassa
product inhibition by PP; Km = 5.4 mM
Code:
r17.2e
Enzyme:
chitin synthase
EC number:
2.4.1.16
Reaction Brenda: UDP-N-acetyl-D-glucosamine
+
[1,4-(N-acetyl-beta-D-glucosaminyl)]n
=>
UDP + [1,4-(N-acetyl-beta-D-glucosaminyl)]n+1
Reversibility:
irreversible
Act & Inh:
Table 24.4
Eff. NAS:
EDTA, Polyoxin D, glcNAc (Aspergillus nidulans), chitodextrin F1 & F2 (Aspergillus flavus),
nikkomycin, digitonin, (Saccharomyces cerevisiae), primulin (Neurospora crassa) and UDP-Nacetyl-D-glucosamine for the organism previously mentioned.
Turnover nr:
unknown
Spec activity:
42.7 μmol/min/mg (Saccharomyces cerevisiae)
Mechanism:
ordered mechanism with UDP-N-acetylglucosamine as the first substrate (Saccharomyces
cerevisiae) [10]
Compartment: cytosol
Table 24.4: Connectivity for chitin synthase
effector
organism
Comment
chit
Saccharomyces cerevisiae
slight inhibition by chitin oligosaccharides
PHser
Neurospora crassa
stimulator
Saccharomyces cerevisiae
stimulator
PHino
Neurospora crassa
stimulator
PHeta
Neurospora crassa
stimulator
UDP
Neurospora crassa
product inhibition; 0.8<Ki<2.2 mM
- 84 -
Saccharomyces cerevisiae
product inhibition; Ki = 2 mM
The overall reaction of r17.2a-e is:
gln + f6p + AcCoA + UTP = glu + chit + HCOA + PP + UDP
Eliminating pyrophosphate with l2 yields
gln + f6p + AcCoA + UTP + H2O = glu + chit + HCOA + 2 Pi + UDP
However this reaction is unequal to r17.2 which does not contain UTP and UDP and produces phosphate instead of
pyrophosphate. Apparently the above discussed biosynthesis of chitine is lumped with the regeneration of UTP:
UDP + Pi => H2O + UTP
Nucleoside-diphosphate kinase (EC 2.7.4.6), which has been discussed for reaction r13.5, catalyzes the regeneration
of UTP by utilizing ATP and UDP as substrates:
UDP + ATP = > UTP + ADP
However due to the utilization of the energy source ATP, the produced ADP should be regenerated. This is
performed by the ATP hydrolyses with has been discussed in r15.1. Eliminating ATP in r13.5 with r15.1 yields the
missing linkage of the regeneration of UTP between the reaction of r17.2 and the overall reaction of r17.2a-e.
24.2
Fructose and mannose metabolism
r17.3: f6P + H + NADH => m1P + NAD
Code:
r17.3
Enzyme:
mannitol-1-phosphate 5-dehydrogenase
EC number:
1.1.1.17
Reaction Brenda: f6P + H + NADH => m1P + NAD
Reversibility:
reversible
Act & Inh:
Table 24.5
Eff. NAS:
adenosine diphosphoribose (Aspergillus niger)
Turnover nr:
unknown
Spec activity:
190 μmol/min/mg (Aspergillus parasiticus), 150 μmol/min/mg (Aspergillus niger), 0.13-0.28
μmol/min/mg (Aspergillus nidulans)
Mechanism:
unknown
Location:
cytosol
Table 24.5: Connectivity for mannitol-1-phosphate 5-dehydrogenase
effector
organism
comment
f6P
Aspergillus niger
Km = 0.54 mM
g6P
Aspergillus niger
competitive inhibitor with respect to f6P
m1P
Aspergillus niger
competitive inhibitor with respect to f6P; Km=0.038
mM
NADH
Aspergillus niger
competitive inhibitor with respect to NAD; Km=0.005
mM
NAD
Aspergillus niger
competitive inhibitor with NADH; Km = 0.083 mM
r17.4: H2O + m1P => man + Pi
Code:
r17.4
Enzyme:
mannitol-1-phosphatase
EC number:
3.1.3.22
Reaction Brenda: H2O + m1P => man + Pi
Reversibility:
irreversible
Act & Inh:
Table 24.6
Eff. NAS:
no effectors
Turnover nr:
unknown
- 85 -
Spec activity:
Mechanism:
Location:
1792 μmol/min/mg (Penicillium notatum, after ammonium sulfate fractionation), 0.16
μmol/min/mg (Neurospora crassa), 0.06 μmol/min/mg (Penicillium islandicum),
0.027 μmol/min/mg (Aspergillus niger), 0.006 μmol/min/mg (Candida utilis).
unknown
cytosol
Table 24.6: Connectivity for mannitol-1-phosphatase
effector
organism
comment
m1P
Aspergillus nidulans
Km = 1 mM
24.3
Starch and sucrose metabolism
r17.5: g6P + H2O + UTP => 2 Pi + UDPglc
This reaction consists of two lumped reactions. No effectors have been identified and therefore the non-zero
elasticities are εr17.5,g6P:cyt, εr17.5,UTP:cyt, εr17.5,Pi:cyt and εr17.5,UDPglc:cyt.
Code:
r17.5a
Enzyme:
phosphoglucomutase
EC number:
5.4.2.2
Reaction Brenda: g6P => g1P
Reversibility:
reversible
Act & Inh:
no entries in the Brenda database.
Eff. NAS:
5,5'-dithiobis(2-nitrobenzoate), p-chloromercuribenzoate, g16P (Km= 0.00224 mM) and g1P
(Km= 0.023 mM) (Saccharomyces cerevisiae)
Turnover nr:
unknown
Spec activity:
205-490 μmol/min/mg (Saccharomyces cerevisiae),
Mechanism:
unknown
Location:
cytosol
Code:
r17.5b
Enzyme:
UTP-glucose-1-phosphate uridylyltransferase
EC number:
2.7.7.9
Reaction Brenda: UTP + g1P = PP + UDPglc
Reversibility:
reversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
The overall reaction of these two intermediate conversions is:
G6P + UTP => PP + UDPglc
Elimination of pyrophosphate with the help of reaction l2 will lead to reaction r17.5.
r17.6: g6P + UDPglc => H + t6P + UDP
Code:
r17.6
Enzyme:
alpha,alpha-trehalose-phosphate synthase (UDP-forming)
EC number:
2.4.1.15
Reaction Brenda: g6P + UDPglc => t6P + UDP
Reversibility:
irreversible
Act & Inh:
Table 24.7
- 86 -
Eff. NAS:
Turnover nr:
Spec activity:
Mechanism:
Location:
ADPglc, UDPglucuronate, TPS-activator protein, UDPglc (Km=0.5 mM) (Saccharomyces
cerevisiae)
unknown
31.48 μmol/min/mg (Neurospora crassa), 15 μmol/min/mg (Saccharomyces cerevisiae), 0.3
μmol/min/mg (Saccharomyces carlsbergensis)
unknown
cytosol
Under conditions of high glycolytic flux, the citrate accumulation is influenced by trehalose-6-phosphate synthase A
in Aspergillus niger.
Table 24.7: Connectivity for trehalose-phosphate synthase
effector
organism
comment
Pi
Saccharomyces cerevisiae
2<Ki<5 mM
Neurospora crassa
inhibitor
AMP
Neurospora crassa
23% inhibition at 10 mM
ADP
Neurospora crassa
slight inhibition
UDP
Neurospora crassa
65% inhibition at 10 mM
UTP
Neurospora crassa
inhibitor
UMP
Neurospora crassa
43% inhibition at 10 mM
g6P
Neurospora crassa
Km = 8.3 mM
UDPglc
Neurospora crassa
Km = 0.8 mM
r17.7: H2O + t6P => Pi + tre
Code:
r17.7
Enzyme:
trehalose-phosphatase
EC number:
3.1.3.12
Reaction Brenda: H2O + t6P => Pi + tre
Reversibility:
irreversible
Act & Inh:
Table 24.8
Eff. NAS:
none found
Turnover nr:
unknown
Spec activity:
unknown
Mechanism:
unknown
Location:
cytosol
Table 24.8: Connectivity for trehalose-phosphatase
effector
organism
comment
Pi
Saccharomyces cerevisiae
activates
24.4
Miscellanous
r17.1: 0.167 ATP + 0.167 g6P + 0.167 H2O => 0.167 ADP + 0.167 H + 0.333 Pi + psacch
This reaction has not been identified in any of the online databases.
r17.8: E4P + H + H2O + NADH => ery + NAD + Pi
This reaction has not been identified in any of the online databases.
25 Biomass formation
- 87 -
r18.17: 0.0342 AApool + 0.00839 chit + 0.000991 ery + 0.00043 ESE + 0.0806 H2O + 0.00246 man + 0.000861
PHchol + 0.000516 PHeta + 0.000344 PHino + 0.422 PROT + 0.282 psacch + 0.0597 RNA + 0.00107 tre +
0.000215 TRIA => biom_cc_gluc
The above mentioned stoichiometric coefficients of the metabolites, which are employed for the formation of the
biomass on glucose, are part of the stoichiometric model of van Gulik et al. The specific growthrate μ is equal to
0.03 h-1.
r18.mu0.05: 0.0344 AApool + 0.0083 chit + 0.00124 ery + 0.000503 ESE + 0.0801 H2O + 0.00247 man +
0.00101 PHchol + 0.000604 PHeta + 0.000403 PHino + 0.42475 PROT + 0.265589 psacch + 0.0514 RNA +
0.00107 tre + 0.000252 TRIA => biom_cc_gluc
The above mentioned stoichiometric coefficients correspond to the biomass formation on a glucose culture with a
specific growrate μ of 0.05 h-1.
26 Penicillin biosynthesis
r19.1: aAd + 6 ATP + cys + 4 H2O + val => ACV + 6 ADP + 6 H + 6 Pi
The enzyme is also influenced by glucose and therefore εr19.1,glc:cyt can be non-zero.
Code:
r19.1
Enzyme:
N-(5-amino-5-carboxypentanoyl)-L-cysteinyl-D-valine synthase
EC number:
6.3.2.26
Reaction Brenda: aAd + cys + val + 3 ATP => ACV + 3 AMP + 3 PP
Reversibility:
irreversible
Act & Inh:
Table 26.1
Eff. NAS:
dithiothreitol (Penicillium chrysogenum)
Turnover nr:
0.133 s-1 (Acremonium chrysogenum)
Spec activity:
unknown
Mechanism:
Michealis-Menten type regarding the individual amino acids and product inhibition by ACV [13]
Location:
cytosol
Table 26.1: Connectivity for N-(5-amino-5-carboxypentanoyl)-L-cysteinyl-D-valine synthase
effector
organism
comment
aAd
Penicillium chrysogenum
Km = 0.045 mM
Acremonium chrysogenum
Km = 0.17 mM
cys
Penicillium chrysogenum
Km = 0.08 mM
Acremonium chrysogenum
Km = 0.026 mM
val
Penicillium chrysogenum
Km = 0.08 mM
Acremonium chrysogenum
Km = 0.34 mM
ACV
Penicillium chrysogenum
product inhibition
ATP
Penicillium chrysogenum
cofactor conc. optimal at 20 mM
glc
Acremonium chrysogenum
inhibition of crude extract due to the deprivation of ATP
via sugar metabolism
Reaction r19.1 can be derived by employing reaction l1 and l2 for the elimination of AMP and PP in reaction r19.1a.
However the reaction then consumes 3 water molecules instead of the required 4. Nevertheless according the the
Brenda database, that 4th water molecule does not take part in the reaction.
r19.2: ACV + O2 => 2 H2O + iPN
No effectors have been identified.
- 88 -
Code:
r19.2
Enzyme:
isopenicillin-N synthase
EC number:
1.21.3.1
Reaction Brenda: ACV + O2 => 2 H2O + iPN
Reversibility:
irreversible
Act & Inh:
Table 26.2
Eff. NAS:
ascorbic acid, bis[H-Cys-D-Val], alpha-aminoadipic(-Cys-Gly) and bis[alpha-aminoadipic(-CysX)] in which X is trp, tyr, phe & valine derivates (Penicillium chrysogenum)
Turnover nr:
4.1 s-1 with ACV as substrate in Aspergillus nidulans
Spec activity:
6.64*10-6 μmol/min/mg (Aspergillus nidulans)
Mechanism:
MM type with respect to ACV, competitive inhibition by glutathione and first order with respect to
oxygen [13].
Location:
cytosol
Table 26.2: Connectivity for isopenicillin-N synthase
effector
organism
comment
O2
Penicillium chrysogenum
activator
Aspergillus nidulans
activator
ACV
Aspergillus nidulans
Km = 0.12 mM
r19.3: 2 ATP + H2O + HCoA + PAA => 2 ADP + 2 H + PAACoA + 2 Pi
No effectors have been identified.
Code:
r19.3
Enzyme:
phenylacetate-CoA ligase
EC number:
6.2.1.30
Reaction Brenda: ATP + PAA + HCoA => AMP + PP + PAACoA
Reversibility:
irreversible
Act & Inh:
Table 26.3
Eff. NAS:
NEM, p-chloromercuribenzoate, 2-mercaptoethanol, dithiothreitol and GSH (Penicillium
chrysogenum)
Turnover nr:
unknown
Spec activity:
0.199 μmol/min/mg (Penicillium chrysogenum)
Mechanism:
unknown
Location:
peroxisome
Table 26.3: Connectivity for phenylacetate-CoA ligase
effector
organism
comment
PAA
Penicillium chrysogenum
Km = 0.0029 mM
Elimation of AMP and PP with the help of l1 and l2 will yield reaction r19.3.
r19.4: H2O + iPN + PAACoA => aAd + HCoA + penG
Code:
r19.4
Enzyme:
isopenicillin-N N-acyltransferase
EC number:
2.3.1.164
Reaction Brenda: PAACoA + iPN + H2O => HCoA + penG + aAd
Reversibility:
irreversible
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Mechanism:
v=k*VAT/(1+KmiPN-PAA/XiPN +KmPAA/XPAACoA ); VAT is the activity of the enzyme involved [13]
Location:
peroxisome
- 89 -
r19.5: aAd => H2O + OPC
This reaction has not been identified in any of the online databases.
r19.6: H + NADPH + O2 + PAA => H2O + NADP + OHPAA
According to KEGG this reaction is spontanous and does not require any catalyst.
r19.7: H2O + iPN => 6APA + aAd
Code:
r19.7
Enzyme:
isopenicillin N amidohydrolase
EC number:
unknown
Reaction Brenda: H2O + iPN => 6APA + aAd
Reversibility:
unknown
Act & Inh:
none found
Eff. NAS:
none found
Turnover nr:
unknown
Mechanism:
v = k*VAT*XiPN/(KmiPN + XiPN); VAT is the activity of the enzyme involved [13]
Location:
peroxisome
r19.8: 6APA + CO2 => 8HPA
The carboxylation of 6APA to 8HPA follows first order kinetics with respect to dissolved CO2 and 6APA according
to Henriksen [17].
r19.9: H2O + PenG => PIO
This reaction has not been found.
27 Transport across the peroxisomal membrane
The stoichiometric model for a glucose strain of Penicillin chrysogenum does not contain the transport relations
r21.8-r21.12. Due to the fact that no specific transport systems are reported of the transport of PAA (r21.1), iPN
(r21.2), OPC (r21.3), 6APA (r21.4), 8HPA (r21.5), aAd (r21.6), penG (r21.7), H2O and CO2, it will be assumed they
migrate across the membrane through passive diffussion. Therefore this phenomenon for any compound i, i.e. any of
the previous mention metabolites, can be described with a simple kinetic law such as vi=klai*(Ci_cyt-Ci_per). Phosphate
(r21.15) is however transported by active transport, i.e. by proton symport. No regulatory mechanism has been found
for the regeneration of ATP in the peroxisome with cytosolic ATP (r21.13). Therefore it will be assumed that all four
reactants are influencing this transport process.
Abbreviations (own)
AIR
= 5-amino-1-(5-phospho-D-ribosyl)imidazole
AMG = 6-aminoglucose
APS
= adenosine 5'-phosphosulfate
AS
= ammonia sulfate
Asucc = argininosuccinate
Bpen = benzylpenicillin
inoh
= Inositol hexakisphosphate
f26P
= fructose 2,6-biphosphasphate
FGAM = 2-(formamido)-N1-(5-phospho-D-ribosyl)acetamidine
GAR = N2-formyl-N1-(5-phospho-D-ribosyl)glycinamide
g1P
= glucose-1-phosphate
g16P = glucose-1,6-diphosphate
- 90 -
MCoA
PAP
PP
Scitr
Ssucc
ThPP
= malonyl-CoA
= phosphoadenosine phosphate
= diphosphate, pyrophosphate
= sodium citrate
= sodium succinate
= thiamin diphosphate
28 References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
Brenda database http://www.brenda.uni-koeln.de
KEGG http://www.kegg.com
Metacyc http://www.metacyc.org/
Sabio Reaction Kinetics database http://sabio.villa-bosch.de/SABIORK/
Patel, S., Martinez-Ripoll, M., Blundell, T.L. & Albert, A. (2002), Structural enzymology of Li(+)sensitive/Mg(2+)-dependent phosphatases, J. Mol. Biol., 320, 1087-1094. [PDF]
Galazzo, J.L. & Bailey, J.E. (1990), Fermentation pathway kinetics and metabolic flux control in suspended
and immobilized Saccaromyces cerevisiae, Enzyme Microb. Technol., 12, 162-172.
Hynne, F., Danø, S. & Sørensen, P.G. (2001), Full-scale model of glycolysis in Saccharomyces cerevisiae,
Biophys. Chem., 94, 121-163.
Teusink, B., Passarge, J., Reijenga, C.A., Esgalhado, E., van der Weijden, C.C., Schepper, M., Walsh, M.C.,
Bakker, B.M., van Dam., K., Westerhof, H.V. & Snoep, J.L. (2000), Can yeast be understood in terms of in
vitro kinetics of the constituent enzymes? Testing biochemistry, Eur. J. Biochem., 267, 5313-5329.
van Gulik, W.M., de Laat, W.T.A.M., Vinke, J.L. & Heijnen, J.J. (2000), Application of metabolic flux
analysis for the udentification of metabolic bottlenecks in the biosynthesis of Penicillin-G, Biotechnology
and Bioengineering, 68(6), 602-618. [PDF]
Faehnrich, M. & Ahlers, J. (1981), Improved assay and mechanism of the reaction catalyzed by the chitin
synthase from Saccharomyces cerevisiae,Eur. J. Biochem., 121(1), 113-118.
Branson, J.P., Nezic, M., Jitrapakdee, S., Wallace, J.C. & Attwood, P.V. (2004), Kinetic characterization of
yeast pyruvate carboxylase isozyme pyc1 and the pyc1 mutant, C249A, Biochemistry, 43 (4), 1075-1081
[PDF]
Bareich, D.C. & Wright, G.D. (2003), Functionally important amino acids in Saccharomyces cerevisiae
aspartate kinase, Biochemical and Bipphysical Research Communinations, 311, 597-603 [PDF]
de Noronha Pissara, P., Nielsen, J. & Bazin, M.J. (1996), Pathway kinetics and metabolic control analysis
of a high-yielding strain of Penicillium chrysogenum during fed-batch cultuvations, Biotechnology and
Bioengineering, 51, 168-176 [PDF]
Hawes, C.S. & Nicolas D.J. (1973), Adenosine 5'-triphosphate sulphurylase from Saccharomyces
cerevisiae, Biochem J., 133(3), 541-550 [PDF]
Seubert, P.A., Hoang, L., Renosto, F. & Segel I.H. (1983), ATP sulfurylase from Penicillium chrysogenum:
measurements of the true specific activity of an enzyme subject to potent product inhibition and a
reassessment of the kinetic mechanism, Arch. Biochem. Biophys., 225(2), 679-691.
Lansdon, E.B., Segel, I.H & Fisher, A.J. (2002), Ligand-induced structural changes in adenosine 5'phosphosulfate kinase from Penicillium chrysogenum, Biochemistry, 41(46), 13672-13680 [PDF].
Henriksen, C.M., Holm, S.S., Schipper, D., Jørgensen, S., Nielsen, J. & Villadsen, J. (1997), Kinectic
studies on the carboxylation of 6-amino-penicillanic acid to 8-hydroxy-penillic acid, Process Biochemistry,
32(2), 85-91 [PDF].
Meixner-Monori, B., Kubicek, C.P., Habison, A.;, Kubicek-Pranz, E.M. & Roehr, M. (1985), Presence and
regulation of the alpha-ketoglutarate dehydrogenase multienzyme complex in the filamentous fungus
Aspergillus niger, J. Bacteriol., 161(1), 265-271 [PDF]
- 91 -
Appendix D: Pool composition matrix
Table D.1: Pool composition matrix
Table D.2: Pool composition matrix with lumped equilibrium pools
Table D. 3: Reduced stoichiometric matrix (PEQ rates, pss metabolites and frozen pools
removed)(41x46)
Table D. 4: Elasticity matrix (46x41)
- 92 -
Appendix E: Transient behaviour unmeasured metabolites
In Figure 0.1 the transient behaviour of unknown metabolites of the glycolysis, the non-oxidative branch
of the PPP and the TCA cycle are shown.
3PG:cyt
GAP:cyt
2.00
1.50
1.00
0.50
-100
0
100
200
300
400
500
-100
2.00
1.50
1.00
0.50
-
0
100
Rib5P:cyt
200
300
400
500
-100
0
2.50
1.50
1.50
2.00
1.00
1.00
0.50
0.50
100
200
300
400
500
-100
1.00
0.50
0
100
200
300
400
500
-100
300
400
500
300
400
500
0
100
200
300
400
500
-100
0
100
OAA:cyt
1.50
500
0.50
200
citr:cyt
1.40
1.20
1.00
0.80
0.60
0.40
0.20
-
2.00
400
1.00
sed7P:cyt
2.50
300
1.50
-
0
200
E4P:cyt
2.00
-100
100
Ribu5P:cyt
2.00
-
-100
Xylu5P:cyt
1.20
1.00
0.80
0.60
0.40
0.20
-
2.50
1.20
1.00
0.80
0.60
0.40
0.20
-
0
100
200
300
400
500
-100
0
100
200
Figure 0.1: Normalized transient behaviour unknown glycolysis, PPP, and TCA metabolites
- 93 -
NADH:cyt
NAD:cyt
4.00
1.00
1.00
1.20
1.00
0.80
0.60
0.40
0.20
-
3.00
2.00
1.00
-100
0
100
200
300
400
500
-100
1.00
1.00
0.99
0.99
0
100
[sec]
200
300
400
500
-100
200
300
400
500
300
400
500
300
400
500
300
400
500
[sec]
FAD:cyt
4.00
1.50
3.00
1.00
2.00
0.50
1.00
-
0
100
200
300
400
500
-100
-
0
100
200
300
400
500
-100
FADH2:cyt
NADPH:cyt
NADP:cyt
1.20
1.00
0.80
0.60
0.40
0.20
-
1.50
1.00
0.50
0.50
-
-
200
300
400
500
-100
0
100
[sec]
200
300
400
500
-100
200
300
400
500
-100
200
[sec]
MYTHF:cyt
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.20
1.00
0.80
0.60
0.40
0.20
-
[sec]
100
METHF:cyt
1.20
1.00
0.80
0.60
0.40
0.20
100
0
[sec]
THF:cyt
0
200
[sec]
2.00
100
100
[sec]
1.00
0
0
[sec]
1.50
-100
100
succCoA:cyt
3.00
2.50
2.00
1.50
1.00
0.50
-
-100
0
[sec]
AcCoA:cyt
-100
HCoA:cyt
0
100
200
[sec]
300
400
500
-100
0
100
200
[sec]
Figure 0.2: Normalized transient behaviour cofactors
- 94 -
Appendix F: Matlab files & In Silico database (part of)
Program to estimate the kinetic parameters for pss metabolites
function [b M]=mregg(Xd,con,ar,lab)
% Multi linear regression, using least squares
%
% Example call: b=mregg(Xd,con,ar,lab)
%
% Fits data to y=b0+b1*x1+b2*x2+ ... bp*xp
% Xd is a data array. Each column of X is a set of data
% Xd(1,:)=x1(:), Xd(2,:)=x2(:), ... Xd(p+1,:)=y(:)
% Xd has n columns corresponding to n data points and p+1 rows
% If con=0, no constant is used, if con~=0 contant term is used
% If ar=0, no analysis of residuals provided
% if ar=~0, analysis of residuals is provided
% lab is a set of user defined lables for explanatory variables;
% Each label mist be 15 characters long, including spaces
% X has n columns corresponding to n data points and p+1 rows
% Function returns vector of coefficients b
% Published in Lindfield, G. & Penny, J., Numerical Methods using Matlab
% Presentice-Hall, Upper Saddle river, 2nd Edition (1999), 310-312.
% Edited by Raymond Blankestijn at 18-02-2007 as original didn't work in
% Matlab 2006b properly
if con==0
cst=0;
else
cst=1;
end
[p1,n]=size(Xd); p=p1-1 ;pc=p+cst;
y=Xd(p1,:)';
if cst==1
w=ones(n,1);
X=[w Xd(1:p,:)'];
else
X=Xd(1:p,:)';
end
% If user lables supplied, these are used, otherwise default labels are
% used
a=zeros(pc,15);
if nargin==3
if cst==1
a(1,1)='Constant
:';
end
for i=1+cst:pc
a(i,:)=strcat('Parameter',numstr(i-cst,2),'
:');
end
else
if cst==1
a=['Constant
:';lab];
else
a=lab;
end
end
% Compute coefficient b, the hat matrix H and the Student t-ratio
C=inv(X'*X);
b=C*X'*y;
H=X*C*X';
SSE=y'*(eye(n)-H)*y;
s_sqd=SSE/(n-pc);
Z=(1/n)*ones(n);
num=y'*(H-Z)*y;
denom=y'*(eye(n)-Z)*y;
R_sqd=num/denom;
Cov=s_sqd*C;
SE=sqrt(diag(Cov));
- 95 -
t=b./SE;
% Compute correlation matrix
V(:,1)=(eye(n)-Z)*y;
for j=1:p
V(:,j+1)=(eye(n)-Z)*X(:,j+cst);
end
SS=V'*V;
D=zeros(p+1, p+1);
for j=1:p+1
D(j,j)=1/sqrt(SS(j,j));
end
Corr_mtrx=D*SS*D;
% Compute VIF
for j=1+cst:pc
ym=X(:,j);
if cst==1
Xm=X(:,[1 2:j-1,j+1:p+1]);
else
Xm=X(:,[1:j-1,j+1:p]);
end
Cm=inv(Xm'*Xm);
Hm=Xm*Cm*Xm';
num=ym'*(Hm-Z)*ym;
denom=ym'*(eye(n)-Z)*ym;
R_sqr(j-cst)=num/denom;
end
VIF=1./(1-R_sqr);
% Print out statistics
if cst==1
fprintf('\nError variance = %8.4f
R_squared= %6.4f\n', s_sqd, R_sqd)
fprintf('\n \n')
fprintf('\n
coeff
SE
t-ratio
VIF\n')
else
fprintf('\nError variance = %8.4f
R_squared= %6.4f\n', s_sqd, R_sqd)
fprintf('\n \n')
fprintf('\n
coeff
SE
t-ratio')
end
if cst==1
fprintf('\n%12s %12.4f %8.4f %10.2f\n', a(1,:), b(1), SE(1), t(1))
end
M=zeros(pc,4);
for j=1+cst:pc
fprintf('\n%12s %12.4f %8.4f %10.2f %10.2f\n', a(j,:), b(j), SE(j), t(j), VIF(j-cst))
M(j,1)=b(j);
M(j,2)=SE(j);
M(j,3)=t(j);
M(j,4)=VIF(j-cst);
%if cst==1
%
fprintf('%8.4',VIF(j-cst))
%end
end
fprintf('\n \n')
% Corr_matrx
% Analysis of residuals
if ar~=0
ee=(eye(n)-H)*y;
s=sqrt(s_sqd);
sr=ee./(s*sqrt(1-diag(H)));
cd=(1/(pc))*(1/s^2)*ee.^2.*(diag(H)./((1-diag(H)).^2));
fprintf('\n
y
Residual
St residual Cook dist.\n')
for i=1:n
fprintf('\n
%12.4f
%10.4f
% 10.4f
%10.4f', y(i), ee(i), sr(i),
cd(i))
end
fprintf('\n \n')
end
Program to calculate the net influx:
function Jin = netfluxin(S,v)
% function which calculates the net flux in of a metabolite pool
- 96 -
% transport across intracellular membranes should be excluded.
[row col]=size(S);
for i=1:row
for j=1:col
if S(i,j)<=0;
S(i,j)=0;
end
end
end
Jin=S*v;
Program to calculate the measured net conversion rates and their corresponding standard deviations.
%Calculation of the rate vector and corresponding covariance matrix for
%growth of P. chrysogenum on glucose
%SYMBOLS USED
%Fuit:
effluent flow
(L/h)
%Fin:
medium feed flow (L/h)
%V:
working volume (L)
%Fgas:
gasflow in (mol/h)
%O2_in:
oxygen concentration in aeration gas (%)
%O2_out: oxygen concentration in exhaust gas (%)
%CO2_in: carbon dioxide concentration in aeration gas (%)
%CO2_out: carbon dioxide concentration in exhaust gas (%)
%Cdw:
biomass concentration (g/L)
%Mw:
Cmol weight (g/Cmol)
%Csubin: glucose feed concentration (mmol/L)
%Cnh4in: ammonium feed concentration (mmol/L)
%Cpain:
PAA feed concentration (mmol/L)
%Csub:
glucose residual concentration (mmol/L)
%Cnh4:
ammonium residual concentration (mmol/L)
%Cpa:
PAA residual concentration (mmol/L)
%Cpeng:
Pen-G concentration (mmol/L)
%Cpio:
PIO concentration (mmol/L)
%Cipn:
IPN concentration (mmol/L)
%C6apa:
6-APA concentration (mmol/L)
%C8hpa:
8-HPA concentration (mmol/L)
%Cohpa:
OH-PAA concentration (mmol/L)
%Copc:
OPC concentration (mmol/L)
%Caa:
amino acids and peptides concentration (mCmol/L)
%Ctoc:
total organic carbon concentration (mCmol/L)
%constants
Vmol=22.474;
C_atomen=6;
%measured flows and concentrations
syms Fuit Fin V Fgas O2_in O2_out CO2_in CO2_out Cdw Mw Csubin Cnh4in Cpain Csub Cnh4 Cpa Cpeng
Cpio Cipn C6apa C8hpa Cohpa Copc Caa Ctoc real
%variances of the measured flows and concentrations
syms var_Fuit var_Fin var_V var_Fgas var_O2_in var_O2_out var_CO2_in var_CO2_out var_Cdw var_Mw
var_Csubin var_Cnh4in var_Cpain var_Csub real
syms var_Cnh4 var_Cpa var_Cpeng var_Cpio var_Cipn var_C6apa var_C8hpa var_Cohpa var_Copc var_Caa
var_Ctoc real
%calculation of the biomass specific conversion rates of S, PAA, NH4, O2,
%CO2, D, Pen-G, PIO, IPN, 6-APA, 8HPA, OH-PAA, OPC, AA & unknown byproducts
%(from the C-balance for the filtrate)
f = [(Fuit*Csub/V-Fin*Csubin/V)/Cdw*Mw;
(Fuit*Cpa/V-Fin*Cpain/V)/Cdw*Mw;
(Fuit*Cnh4/V-Fin*Cnh4in/V)/Cdw*Mw;
Fgas*((100-O2_in-CO2_in)/(100-O2_out-CO2_out)*O2_out-O2_in)/(V*Cdw)*Mw*10;
Fgas*((100-O2_in-CO2_in)/(100-O2_out-CO2_out)*CO2_out-CO2_in)/(V*Cdw)*Mw*10;
Fuit/V*1000;
Fuit*Cpeng/(V*Cdw)*Mw;
Fuit*Cpio/(V*Cdw)*Mw;
Fuit*Cipn/(V*Cdw)*Mw;
Fuit*C6apa/(V*Cdw)*Mw;
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Fuit*C8hpa/(V*Cdw)*Mw;
Fuit*Cohpa/(V*Cdw)*Mw;
Fuit*Copc/(V*Cdw)*Mw;
Fuit*Caa/(V*Cdw)*Mw;
Fuit*(Ctoc-C_atomen*Csub-8*Cpa-16*Cpeng-16*Cpio-14*Cipn-8*C6apa-9*C8hpa-8*Cohpa-6*CopcCaa)/(V*Cdw)*Mw];
%calculation of the covariance matrix
v=[Fuit,Fin,V,Fgas,O2_in,O2_out,CO2_in,CO2_out,Cdw,Mw,Csubin,Cnh4in,Cpain,Csub,Cnh4,Cpa,Cpeng,Cpi
o,Cipn,C6apa,C8hpa,Cohpa,Copc,Caa,Ctoc];
R = jacobian(f,v);
vars = [var_Fuit var_Fin var_V var_Fgas var_O2_in var_O2_out var_CO2_in var_CO2_out var_Cdw
var_Mw var_Csubin var_Cnh4in var_Cpain var_Csub var_Cnh4 var_Cpa var_Cpeng var_Cpio var_Cipn
var_C6apa var_C8hpa var_Cohpa var_Copc var_Caa var_Ctoc];
B = diag(vars);
COV = R*B*R';
%substitution of the measurements and their variances
Fuit=data(1);
Fin=data(2);
V=data(3);
Fgas=data(4);
O2_in=data(5);
O2_out=data(6);
CO2_in=data(7);
CO2_out=data(8);
Cdw=data(9);
Mw=data(10);
Csubin=data(11);
Cnh4in=data(12);
Cpain=data(13);
Csub=data(14);
Cnh4=data(15);
Cpa=data(16);
Cpeng=data(17);
Cpio=data(18);
Cipn=data(19);
C6apa=data(20);
C8hpa=data(21);
Cohpa=data(22);
Copc=data(23);
Caa=data(24);
Ctoc=data(25);
var_Fuit=variances(1);
var_Fin=variances(2);
var_V=variances(3);
var_Fgas=variances(4);
var_O2_in=variances(5);
var_O2_out=variances(6);
var_CO2_in=variances(7);
var_CO2_out=variances(8);
var_Cdw=variances(9);
var_Mw=variances(10);
var_Csubin=variances(11);
var_Cnh4in=variances(12);
var_Cpain=variances(13);
var_Csub=variances(14);
var_Cnh4=variances(15);
var_Cpa=variances(16);
var_Cpeng=variances(17);
var_Cpio=variances(18);
var_Cipn=variances(19);
var_C6apa=variances(20);
var_C8hpa=variances(21);
var_Cohpa=variances(22);
var_Copc=variances(23);
var_Caa=variances(24);
var_Ctoc=variances(25);
%calculation of the rate vector, associated covarance matrix and variance
- 98 -
%vector
rates=subs(f);
varcovar=subs(COV);
var=diag(varcovar);
rates
varcovar
var
Program to calculate balanced net conversion rates
function [rm_est Prm_est h Pe] = rmest (Rind,Prm,rm)
% [rm_est Prm_est h] = balrm (Rind,Prm,rm)
% This function balances the measured conversion rates, which requires
% reduced redundancy matrix (Rind), covariance matrix of the measurements
% (Prm) and the measured conversion rates (rm).
% Output are the estimates on the measured conversion rates (rm_est), the
% covariance matrix of the estimates (Pr_est) and the test function (h).
% The redundancy matrix can be calculated by the [R Ecpi]=Rpi(Em,Ec)
% command
Pe=Rind*Prm*Rind';
% Estimation of measured conversion rates
Const=Prm*Rind'*inv(Pe)*Rind;
[r k]=size(Const);
rm_est=(eye(r,k)-Const)*rm;
Prm_est=Prm-Prm*Rind'*inv(Pe)*Rind*Prm;
% test function h
epsilon= Rind*rm;
h=epsilon'*inv(Pe)*epsilon;
function [rc_est Prc_est]=rcest(rm_est, Prm_est, Ecpi, Em)
% [rc_est Prc_est]=rcest(rm_est, Prm_est, Ecpi, Em)
% This function calculates the balanced non-measured conversion rates
% (rc_est) and its covariance matrix (Prc_est). It requires the estimates
% of the balanced measured conversion rates (rm_est), its covariance matrix
% of (Prm_est) the pseudo inverse of the elementary composition matrix of
% the non-measured rates (Ecpi) and the elementary composition matrix of
% the measured coversion rates (Em) as input.
% see also rmest.m and Rpi.m for the calculation of these inputs.
% Estimation of non-measured conversion rates
rc_est=-Ecpi*Em*rm_est;
Prc_est=Ecpi*Em*Prm_est*Em'*(Ecpi)';
function [R Ecpi] = Rpi(Em, Ec)
% calculation of pseudo inverse matrix R
[U S V]=svd(Ec'*Ec);
n=length(S);
Spi=zeros(n,n);
for i = 1:n
if S(i,i)==0
Spi(i,i)=0;
else
Spi(i,i)=1/S(i,i);
end
end
Ecpi=(V*Spi*U')*Ec';
R=Em-Ec*Ecpi*Em;
Program to help lumping reaction manually in stoichiometric model:
function nonzero=lumping(S,x,v)
% lumping reactions with the help of stoichiometric matrix S and specifying
% the reactions to be lumped in vector x
[row col]=size(S);
vec=zeros(row,1);
for i=1:length(x);
- 99 -
vec=vec+S(:,x(i))*v(i);
end
[r s t]=find(vec);
nonzero = [r t];
Finding zero columns or rows:
function zerorows = zeroentriesrows(S)
% finding columns with zero entries
zerorows=[];
for i=1:length(S(:,1))
x=sum(abs(S(i,:)));
zerorows(i,1)=i;
zerorows(i,2)=x;
end
Calculating number of non-zero elasticities in columns
function M = sumcheck(ex)
% calculates the number of non-zero elasticities in the columns
% Delft 21st of September
M=zeros(length(ex(1,:)),2);
for i=1:length(ex(1,:))
M2=size(find(ex(:,i)));
M(i,2)=M2(1);
M(i,1)=i;
end
M;
Part of In Silico database3:
**** HEADER ****
Type: Transformer
Setup: Block
Version: 1.4
Name: Penicillium chrysogenum (DS12975) Forza Inter
Last change: 27.12.06
**** EOF HEADER ****
**** Reaction ****
Name:
Short name:
R1
r1.1
Systematic name:
EC:
KEGG:
Reaction:
ATP + glc => ADP + g6P + H
Location:
cytosol
Pathway:
glycolysis
**** EOF Reaction ****
**** Transport ****
Name:
Short name:
R35
r6.1
3
For explanotary reason only a reaction and a transport are shown. The In Silico database contains all reactions of
the pen G model and all lumped reactions and is too large to include in the report
- 100 -
Systematic name:
EC:
KEGG:
Reaction:
11*H_mitochondria + NADH_mitochondria + 0.5*O2_cytosol => 10*H_cytosol + H2O_cytosol + NAD_mitochondria
Location:
mitochondria
Pathway:
oxidative phosphorylation
**** EOF Transport ****
- 101 -
- 102 -