Introduction to Metabolism
After this lecture you will
be able to answer:
• What are the biological roles of ATP?
• How is ATP formed?
• What are the similiarities and differences
between oxidation reduction reactions
mediated by NAD vs FAD?
• What are the properties of metabolic
pathways?
• Why are metabolic Pathways regulated?
• How can one elucidate the order of metabolic
pathways, and how they are regulated?
Metabolism
(The Acquisition and Utilization of Free Energy)
exergonic
endergonic
Carbon Atom Oxidation
Reduced

Oxidized
CH2 —> CH2OH —> C=O —> COOH —> CO2
Lipids
Glucose
Roles of ATP and NAD(P)+
in Metabolism
ATP
Triphosphate
Adenine
Ribose
Hydrolysis of ATP
Phosphate Compounds
Roles of ATP
(Coupled Reactions)
• Provides energy for endergonic processes
• Biosynthesis, Mechanical Work, Active Transport
• Early stages of nutrient breakdown
– Jump start Catabolism
Fructose-6-P + ATP ——> Fructose-1,6-bP + ADP
∆Go’
(kJ/mol)
---------Fructose-6-P + Pi ——> Fructose-1,6-bisP + H2O
+13.3
ATP + H2O ——> ADP + Pi
-30.5
------------------------------------------------------------------------------Fructose-6-P + ATP ——> Fructose-1,6-bisP + ADP
-17.2
Roles of ATP
• Interconversion of nucleoside triphosphates
NDP + ATP ——> NTP + ADP
Nucleoside Diphosphate Kinase
• Additional phosphoanhydride cleavages in
highly endergonic reactions
(NMP)n + NTP ——> (NMP)n+1 + PPi
PPi + H2O ——> 2 Pi
Pyrophosphatase
Sources of ATP
Phototrophs: photosynthesis
Chemotrophs: oxidation of organic
compounds (e.g. carbohydrates, lipids,
and proteins)
Formation of ATP
• Substrate-level phosphorylation
X–P + ADP ——> X–H + ATP
• Oxidative phosphorylation
• Photophosphorylation
• Adenylate Kinase reaction
2 ADP ——> AMP + ATP
Substrate-Level Phosphorylation
Oxidative Phosphorylation
Photophosphorylation
Source of NAD(P)+, and other cofactors
NADP+
Nicotinamide Adenine Dinucleotide (Phosphate)
O
C
O
P O CH2
O–
O
N
O
OH
O
P O CH2
O–
OH
NH2
Nucleotide
OH
O
A
OPO3=
Nucleotide
Niacin
Figure 14-1
Reduction of NAD(P)+ to NAD(P)H
2 e- transfer
Figure 14-11
Oxidation-Reduction Reactions
SH2 + NAD+ ——> S + NADH + H+
SH2 + FAD ——> S + FADH2
SH2: Reduced Substrate
S: Oxidized Product
NAD+: Electron Acceptor
FAD: Electron Acceptor
Flavin Adenine Dinucleotide (FAD)
Figure 14-12
Reduction of FAD to FADH2
2 e- transfer, sequentially
Figure 14-13 part 1
Reduction of FAD to FADH2
2 e- transfer, sequentially
Figure 14-13 part 2
Reduction of Protein bound Metals
1 e- transfer
Fe3+ + Cu+
Electron Electron
Acceptor Donor
Fe2+ + Cu2+
Half-Reactions
Fe3+ + e–
Cu+
Oxidation
Involves
Loss
Fe2+
(reduction)
Cu2+ + e–
(oxidation)
(e- or H:-)
Reduction
Involves
Gain
• Each half reaction has a Reduction Potential (E)
– Affinity for electrons; Higher E, Higher Affinity
– Electrons transferred from lower to higher E
Standard Reduction Potentials
Standard Reduction Potential
Difference
∆Eo’ = Eo’(e– acceptor) – Eo’(e– donor)
∆Go’ = –nF∆Eo’
For negative G need positive E
E(acceptor) > E(donor)
Note: reduction potential is extremely pH sensitive
E = Eo’ + 0.06V*(7-pH)
Which of the following statements concerning ATP is
FALSE?
A. Although they are sometimes called "high energy"
bonds, the phosphoanhydride bonds in ATP are no
different from other covalent bonds.
B. The cleavage of phosphoanhydride bonds in ATP
releases large amounts of free energy.
C. Phosphate groups within ATP experience more
resonance stabilization than free phosphate groups.
D. The cleavage of phosphoanhydride bonds in ATP
relieves electrostatic repulsion.
While ____ is always involved in reactions that
require the transfer of 2 electrons, ____ can
participate in reactions that transfer either 1 or 2
electrons.
A)
B)
C)
D)
E)
O2; NAD+
NAD+; FAD
NAD+; O2
flavin; niacin
FAD; NAD+
Metabolic Pathways
Metabolites
(Intermediates)
A ——> B ——> C ——> D ——> E
1
2
3
4
Enzymes
Metabolic
Map
Overview of
Catabolism
Figure 14-3
Properties of Metabolic Pathways
• Separate Anabolic and Catabolic Pathways
• Steady-State
• Irreversible (overall): reversibility of individual steps
• First Committed (Exergonic) Step: others close to
equilibrium
• Compartmentation (organelles & tissues): isoenzymes
and transport
• Regulation (usually first committed step): often
rate-limiting
Separate Anabolic and Catabolic Pathways
Potential Futile Cycles
(Regulation)
Biosynthesis
Central
Metabolite
Product/Nutrient
Catabolism
Steady State
Steady-State
A
Input
A
B
Output
B
Thermodynamics of individual steps
A  B
Go’ = -RTlnKeq
Not standard conditions or at equilibrium:
G = Go’+RTln([B]/[A])
Three Physiological Conditions:
Go’<<<<<<0 : G always negative
Example: ATP hydrolysis
Go’>0 : near equilibrium, reversible,
direction depends on actual [B]/[A]
Example: Most reactions
Go’>>>>>>0 : G always positive, must be coupled
Example: Phosphorylation of Glucose
Go’>0
Go’>0
Go’>0
Go’>0
Go’>0
Go’>0
Which of the following statements concerning
metabolic pathways is FALSE?
A. Pathways are directional.
B. Metabolic intermediates are maintained in a steady
state.
C. Opposing pathways may share several reversible
reactions.
D. Metabolic pathways may have either a net positive
or net negative free energy change.
Regulation of Biosynthetic
Pathways
Rationale for Regulation
Biosynthesis
Macromolecules
Central
Metabolite
Product
(e.g. Amino Acid)
Catabolism
Nutrient
Efficiency and Flexibility
Biological Efficiency
• Biosynthesis
– Synthesize precursors not available in diet
– Cease synthesis when precursors become
available in diet (pre-existing enzymes)
– Produce precursors and macromolecules at
appropriate rates
• Catabolism
– Degrade most appropriate nutrients at
appropriate rates
Biological Flexibility
• Adaptaton to Dietary Changes
– Need for biosynthetic products
– Catabolism of new nutrients
– Control of pre-existing enzymes
• Metabolic Flux
– Rates of metabolism reflecting needs
for energy and macromolecular synthesis
Competing Reactions: Regulation
A
Enzyme 1
B
Enzyme 2
C
Control Mechanisms
• Control of Enzyme Availability
– Induction/Repression
• Control of Enzyme Activity
– Covalent/Non-covalent
• Control of Substrate Availability
Principles Governing Controls of
Enzyme Catalytic Activity
• Regulated Enzymes
– Enzyme catalyzing committed, rate-limiting
step (often first step)
– Thermodynamically highly favorable reaction
• Outcomes of Regulation
– Modulation of metabolic flux
Types of Regulation
• Specific: pathway’s substrate or product
• General: needs for C or N sources or
growth rates (e.g. energy charge)
Specific Controls
• Control of Enzyme Amount
– Modulation of gene expression in response
to a specific molecule
• Cholesterol repression
• Lactose induction
• Control of Enzyme Activity
– In response to Pathway specific metabolites
• Fructose-1,6-bisphosphate/Citrate
General Controls
(Integration of Cellular or Organism Functions)
• Internal Effectors
– Catabolite Repression (Glucose)
– Energy Charge (ATP/ADP/AMP)
– Reduction Potential (NAD+/NADH)
• External Effectors (e.g. hormones)
Significance: Efficiency and Flexibility!
Signals Mediating Regulation
Availability of
Substrates or Products
(Ligands)
Regulatory Proteins
Biosynthetic Pathways
ATP
Central
Metabolite
ADP + Pi
Product
(Amino Acid)
Simple Feedback Inhibition
Central
Metabolite
Product
(Amino Acid)
X
ATP
ADP + Pi
Complex Feedback Inhibition
Central
Metabolite
X
X
X
Product 1
Product 2
Mechanisms of
Complex Feedback Inhibition
• Cumulative: sum of individual inhibitions
• Concerted: both end products required for
inhibition
• Isoenzymes: two enzymes, each inhibitable
by a different end product
Cumulative Feedback Inhibition
D
A
B
E
C
A
F
A
B
B
G
D
E
F
G
C
D
E
F
G
C
Concerted Feedback Inhibition
D
A
B
E
C
A
F
A
B
B
G
D
E
F
G
C
D
E
F
G
C
Isozymes
1
A
D
B
1
E
C
A
2
F
A
B
2
2
G
1
B
D
E
F
G
C
D
E
F
G
C
Modulation of Metabolic Flux
Energy Charge
Energy Charge
(Daniel Atkinson)
Energy Charge =
1
2
2ATP + ADP
ATP + ADP + AMP
Degree of Phosphorylation of the
Adenylate Pool
Steady-State E.C. = 0.93
ATP, ADP and AMP = Regulatory Ligands
Energy Charge
Anabolic pathways
(Biosynthesis)
Catabolic Pathways
• ATP Utilizing
• ATP Regenerating
• Activated
• Activated
– High EC (ATP)
• Inhibited
– Low EC (AMP)
(Degradation)
– Low EC (AMP)
• Inhibited
– High EC (ATP)
Activity of Regulated Enzymes in
ATP Utilizing vs Regenerating pathways
100
80
% of 60
Vmax
40
R-type
E.C in the Cell
around 0.9
U-type
20
0
0
0.2
0.4 0.6 0.8
Energy Charge
1
Cells control or regulate the flux through metabolic
pathways by means of
I. allosteric control of enzymes.
II. covalent modification of enzymes.
III. genetic control of the concentrations of enzymes.
IV. genetic expression of allosteric effectors.
A)
B)
C)
D)
E)
I, II, III, IV
II, III
I, II, IV
I, II, III
I, IV
Negative feedback inhibition is one type of
_______ metabolic control.
A)
B)
C)
D)
non-covalent
hormonal
covalent
genetic
Experimental Approaches
to Metabolism
Features of Metabolic Pathways
A ——> B ——> C ——> D ——> E
(1) Sequences and Energetics
(2) Enzymes and Mechanisms
(3) Control Mechanisms (Regulation)
(4) Compartmentation
Elucidation of Metabolic Pathways
A ——> B ——> C ——> D ——> E
Metabolic Inhibitors: accumulation
of intermediates
Biochemical Genetics: mutants
Pathway Labeling: isotopes
Metabolic Inhibitors
(Accumulation of Intermediates)
(e.g. Glycolysis)
A ——> B ——> C ——> D ——> E
Example: Inhibition of Enolase by Fluoride:
-results in accumulation of 2-phosphoglycerate and
3–phosphoglycerate)
Biochemical Genetics
(Mutants)
Natural Genetic Defects
Manipulation of Microorganisms
Accumulation of Intermediates
A ——> B ——> C ——> D ——> E
Growth Requirements (auxotrophic mutants)
A ——> B ——> C ——> D ——> E
Pathway Labeling
A* ——> B* ——> C*
Stable Isotopes
Radioisotopes
Detection of Isotopes
• Stable Isotopes
– Mass Spectrometry
– NMR
• Radioisotopes
– Proportional Counting (Geiger Counter)
– Liquid Scintillation Counting
– Autoradiography
Quantify Differential Expression
Condition 1
Condition 2
Sample Prep
Sample Prep
Mix samples
and detect
Quantify Differences
Quantify Differential Expression
• Transcription (RNA):
– Microarray
• Proteomics (Protein):
– 2D-SDS-PAGE
– Isotope Coded Affinity Tag
Microarray
2D-SDS-PAGE
ICAT Chemistry
ICAT = Isotope Coded Affinity Tag
LIGHT
IAM
Reactive
Isotope
Group
(specific for code
13C)
(D
or
cysteines)
HEAVY
IAM
Biotin
Affinity
Tag
Biotin
Same
behavior
chemically,
but different
in mass.
Protein samples from cells grown under two different conditions
Condition 1
Condition 2
SH
SH
SH
SH
Sample 1
labeled with
Light ICAT
+
S
+
Light
Light
S
S
Heavy
Sample 2
labeled with
Heavy ICAT
S Heavy
Light
Light
Samples mixed together
Trypsin digest
Peptide purification
Liquid Chromatography 
MS(for peaks)/MS(for identification)
______ is the most reliable systems biology
approach to assess gene expression.
A)
B)
C)
D)
E)
genomics
transcriptomics
proteomics
metabolmics
none of the above
• What are the biological roles of ATP?
• How is ATP formed?
• What are the similiarities and differences
between oxidation reduction reactions
mediated by NAD vs FAD?
• What are the properties of metabolic
pathways?
• Why are metabolic Pathways regulated?
• How can one elucidate the order of metabolic
pathways, and how they are regulated?
You analyze 4 mutants (1,2,3,4) that have defects in the four
enzymes of a biosynthetic pathway for the accumulation of
intermediates (A,B,C,D,E). You obtain the following data:
A
B
C
D
E
1
-
+
+
-
+
2
-
+
-
-
-
3
+
+
+
-
+
4
-
+
-
-
+
Indicate the order of the intermediates in the pathway and the
enzyme that catalyze each step.
You analyze 4 mutants (1,2,3,4) that have defects in the four
enzymes of a biosynthetic pathway for B, for their ability to grow
on intermediates (A,B,C,D,E). You obtain the following data:
A
B
C
D
E
1
-
+
+
-
+
2
-
+
-
-
-
3
+
+
+
-
+
4
-
+
-
-
+
Indicate the order of the intermediates in the pathway and the
enzyme that catalyze each step.
You grow yeast media containing 2% or 0.1% glucose.
Samples containing high glucose are modified with Light
ICAT, and low glucose samples are modified with Heavy
ICAT. After analysis of the samples you identify a protein with
the MS spectra below.
What can you conclude about the regulation of the expression
of this protein?