Chapter 3 Enzyme1
introduction to enzymes
Lecture Outline
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Introduction to enzyme
Properties and catalytic mechanisms of
enzymes
Structure and function of enzymes
Nomenclature and classification of enzyme
Kinetics of enzyme-catalyzed reaction
Regulation of enzyme activity
Clinical application of enzymes
Essential Question:
What are enzymes, and what do they do?

Living Cells: Thousands of chemical reactions are
proceeding very rapidly at any given instant.

All reactions are catalyzed by special biocatalysts,
enzymes —protein and occasionally RNA.

Reactions in cells are different from general chemical
reactions: easily, rapidly regulated and controlled

Enzyme-catalyzed reaction take place usually under
relatively mild conditions.
What do enzymes do?

Enzymes are biological catalysts that
accelerate the rates of chemical
reactions.
Snail without
enzyme catalyst
Snail with enzyme catalyst
What is the difference between an
enzyme and a protein?
Protein
Enzymes
RNA
All enzymes are
proteins except
some RNAs and
not all proteins
are enzymes
Substrates, products, and enzymes
Enzymes catalyze the rate at which
substrates are converted to product
Enzyme
Substrates
Product
Enzymes catalyze the conversion of
substrates into products

What is a substrate?
 A substrate is the compound that is converted
into the product in an enzyme catalyzed reaction.
 For the reaction catalyzed by aldolase, fructose
1,6-phosphate is the substrate.
What is the difference between
enzyme catalyzed reactions and
uncatalyzed chemical reactions?
Enzyme catalyzed reactions are much
faster than uncatalyzed reactions.
 Enzyme catalyzed reactions display
saturation kinetics with respect to
substrate concentration.
 Enzyme catalyzed reactions are optimized
for specific temperature and pH values.

Molecular components
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Simple enzymes: enzymes require no other
chemical groups other than their amino acid
residues for activity. consists of only one peptide
chain.
trypsin, chymotrypsin, ribonuclease A
Conjugated enzymes: enzymes contain
chemical groups other than AA, the non-amino
acid parts are usually called cofactors
the protein part alone called apoprotein
(apoenzyme).
holoenzyme= apoenzyme (pr) +cofactor (non-pr)
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Many vitamins, organic nutrients required
in small amounts in the diet, are
precursors of cofactors.
Cofactors: metal ions, small molecules,
Cofactors are divided two groups according
to the binding ability.
----coenzyme
-----prosthetic group
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Coenzymes:
Loosely bind to apoenzyme. Be able to be
separated with dialysis
Accepting H+ or group and leaving to transfer
it to others, or vise versa.
Prosthetic groups:
Tightly bind through either covalent or many
non-covalent interactions.
Remained bound to the apoenzyme during
course of reaction.
Metal ions

Metal-activated enzyme: ions necessary but
loosely bound. Often found in metal-activated
enzyme. Metal ion is essential for activity

Metalloenzymes: ions tightly bound
Particularly in the active center, transfer
electrons, bridge the enzyme and substrates,
stabilize enzyme conformation, neutralize the
anions.
Metal ion is retained throughout purification
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Organic componds
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Small size and chemically stable compounds
Transferring electrons, protons and other groups
Vitamin-like or vitamin-containing molecule
Coenzymes often function as transient carriers
of specific (functional) groups during catalysis.
Monomeric\oligomeric\multienzy
me enzymes
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Monomeric enzyme: only contain a polypeptide
chain with tertiary structure.
Oligomeric enzyme: contain two or more
polypeptide chains associated by noncovalent
forces.
Multienzyme complex: different enzymes catalyzed
sequential reactions in the same pathway are
bound together.
Multifunctional enzyme(tandem enzyme): a single
polypeptide chain with multiple activities
Components of enzyme
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Essential groups: all groups essential for
maintaining the enzyme activity
active center:
a specific region of the enzyme that contains
some chemical groups for binding substrates
and transforming it into products.( some
functional groups are close enough in space to
form a portion)
The active site is a three-dimensional
conformation formed by groups that come
from different parts of the linear amino acid
sequence. Look like a cleft or a crevice.
hydrophobic.
Two essential groups
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Active site contains
binding groups (specificity of binding): to
associate with the reactants to form an
enzyme-substrate complex
determine specificity for substrate
catalytic groups (participate in the catalytic
processes) :to catalyze the reactions and
convert substrates into products
determine characteristics of catalyzation
Maintain conformation of enzyme
Other essential
groups
Substrate
Ser: OH
Cys: SH
His: imidazole
Binding
groups
Catalytic
groups
Active site
Essential groups of the active site

In many enzymes, the active site has
shape complementary to those of their
substrates only after the substrates are
bound (the induced fit).

For conjugated enzymes, the active site
always contains coenzymes.
Two models have been
proposed for substrate
and enzyme binding:
(1) Lock and key model
(2) Induced fit model

Emil Ficher: (1894)

The ‘Lock & Key’ hypothesis
-
explains substrate specificity
- says nothing about why catalysis occurs

Dan Koshland: (1958)
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‘Induced Fit’ hypothesis:
-
enzymes prefer to bind to a distortion of the
substrate that resembles the transition state
-
both enzyme and substrate must adjust to one
another
 - in reality, enzyme is not ‘distorted’ but has
evolved to bind in a certain way and sometimes
undergo conformational changes
Lock and key model
Induced fit model
An Example: Induced conformational
change in hexokinase
Catalyzes phosphorylation of glucose to glucose 6-phosphate during
glycolysis
such a large change in a protein’s conformation is not unusual
BUT: not all enzymes undergo such large changes in
conformation
Advantage of the induced fit mechanism

The active site can be open to allow substrates to
bind, then close over the substrates to provide
optimum transition state stabilization
Disadvantage of the induced fit mechanism
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
Energy that would otherwise be used to help
stabilize the transition state of the reaction
Energy must be used to induce the
conformational change in the enzyme.
Notice that in both the Lock
and Key and Induced Fit
models, the active site is
designed to stabilize the
transition state of the
reaction.
Several factors contribute to
enzyme catalysis
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Proximity and orientation effects
Electrostatic effects
Acid-base catalysis
Covalent catalysis
What characteristic features define
enzymes?

Enzymes are remarkably versatile biochemical
catalysts that have in common three distinctive
features: catalytic power, specificity, and
regulation

Common features: 2 “do” and 2 “don’t
Unique features: 3 “high”

Common features
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Do not consume themselves: no changes in
quantity and quality before and after the reactions
Do not change the equilibrium points: only
enhance the reaction rates.
Apply to the thermodynamically allowable
reactions
Reduce the activation energy
Unique features

Enzyme-catalyzed reactions have very high
catalytic efficiency.

Enzyme have a high degree of specificity for
their substrates.

Enzymatic activities are highly regulated in
response to the external changes.
High Catalytic power:
the ratio of E-catalyzed rate of a reaction
to the uncatalyzed rate

Enzyme display
enormous catalytic
power, accelerating
reaction rates as much
as 1016 over uncatalyzed
levels.
Urease is a good example:
Catalyzed rate: 3x104/sec
Uncatalyzed rate: 3x10 -10 /sec
Ratio is 1x1014 !
High Specificity: refers to the ability of
an enzyme to discriminate between two
competing substrates and catalyze one
specific reaction
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Specificity: the selective qualities of an enzyme
(selecting substrate)
Based on structural complementarity, enzyme
recognize substrate.
Unlike conventional catalysts, enzymes
demonstrate the ability to distinguish different
substrates.
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Enzymes are highly specific both in the
reaction catalyzed and in their choice of
substrates.
An enzyme usually catalyzes a single chemical
reaction or a set of closely related reactions.
There are three types of substrate specificities.
Absolute specificity
Relative specificity
Stereospecificity
Absolute specificity

Enzymes can recognize only one type of substrate
and implement their catalytic functions
. For example,
 urease only hydrolyzes urea to form NH3 and CO2.
 not methylurea.
Relative specificity

Enzymes catalyze one class of substrates or
one kind of chemical bond in the same type

Protein kinase A ,C,G, To phospharylate the –
OH group of serine and threonine in the
substrate proteins, leading to the activation of
proteins.
stereospecificity

The enzyme can act on only one form of
isomers of the substrates.

Lactate dehydrogenase can recognize only
the L-form but the D-form lactate.
High regulation

Enzyme-catalyzed reactions can be regulated in
response to the external stimuli, satisfying the
needs of biological processes

Regulation can be accomplished through varying
the enzyme quantity, adjusting the enzymatic
activity, or changing the substrate concentration.
Classification of enzyme
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1956, International Commission on Enzymes (IUBMB)

-Classified by the types of reactions that they catalyze
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6 classes recognize
Oxidoreductases, Transferases, Hydrolases,
Lyases, Isomerases, Ligases
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With each class subdivided into further subclasses
(1) Oxidoreductase
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Catalyze oxidation-reduction reactions.
catalyze the transfer of hydrogen atoms and
electrons
Contains dehydrogenase and oxidase.
For example: lactate dehydrogenase, peroxidase.
(2) Transferase
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Catalyze transfers of groups between donors and
acceptors.

For example: glutamate-pyruvate trasaminase (GPT),
acyltransferase.
(3) Hydrolase
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Catalyze cleavage of bonds by addition of water.
For example: pyrophosphatase, peptidase.
(4) Lyase
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Catalyze lysis of a substrate, generating a double bond
or adding a substrate to a double bond of a second
substrate.
For example: pyruvate decarboxylase, aldolase.
(5) Isomerase
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Catalyze racemization of optical or geometric isomers
and certain intramolecular oxidation-reduction
reactions.
For example: alanine racemase, mutase.
(6) Ligase or Synthetase
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Join two molecules at the expense of a high energy
phosphate bond of ATP.
For example: glutamine synthetase, carboxylase.
Enzyme nomenclature
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Traditional nomenclature:

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the suffix –ase : urease, phosphatase,
some bearing little resemblance to their activity:
trypsin and pepsin (proteases) — easy confuse
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Systematic nomenclature:

Each enzyme is given a systematic name
which identifies the reaction catalyzed.
Hexokinase----ATP: D-hexose-6phosphotrasferase
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A four digital number can precisely identify all
enzymes
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Each enzyme is assigned a four-digit number
with the first digit denoting the class it belongs,
the other three further clarifications on the
reaction catalyzed.
Hexokinase: E.C. 2.7.1.1

class 2, transferase
Systematic name
Substrates are stated first, followed by the reaction type
to which the ending-ase is affixed.
Serial number: EC. X.X.X.X
（ EC——Enzymes Commission ）
Lactate:NAD+ oxidoreductase
EC
1.
1.
1. 27
Major class----oxidoreductase
Subclass----oxidation group CHOH
Subsubclass----NAD+ as hydrogen acceptor
No. in this subsubclass
Common name: Lactate dehydrogenase (LDH)
Some definitions
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Apoenzyme = the protein part of an enzyme without
coenzymes or prosthetic groups that are required for the
enzyme to have activity. (Note: many enzymes do not have
coenzymes or prosthetic groups bound to them).
Coenzyme = small organic or inorganic molecules which are
bound to the apoenzyme and are required for the enzyme to
catalyze the chemical reaction.
Prosthetic group = similar to a coenzyme, but is tightly bound
to the apoenzyme. Heme is a prosthetic group in cytochrome
c and hemoglobin.
Holoenzyme = the apoenzyme with the coenzyme or
prosthetic group bound to it (i.e. the active form of the
enzyme).
Enzyme 2
kinetics
Kinetics：is a study on the rate of
enzyme-catalyzed reactions and the
factors affected the reaction rate.
The factors affected the rate of enzymecatalyzed reactions：
substrate
pH
enzyme
activator
temperature
inhibitor
For a reaction to occur, the substrates
must first overcome an energy barrier.
The higher the energy barrier, the
slower the reaction will occur..
For most of the reactions in
our bodies, the substrates
must overcome large energy
barriers for the reaction to
occur. This prevents us
from spontaneously bursting
into flames. Enzymes lower
the energy barrier for a
reaction, speeding up the
reaction in a controlled
fashion and preventing
undesirable side reactions
from occurring.
Enzymes catalyze reactions by
lowering their activation energies
Free Energy (DG)
..
Substrates
Uncatalyzed
Enzymes
Reaction
stabilize
Enzyme the
Catalyzed transition
Reaction states of
reactions
DG rxn
Products
Reaction Coordinate
Intermediate state
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Forming an enzyme-substrate complex, a
transition state, is a key step in the catalytic
reaction
Transition state is a fleeting molecular moment
(not a chemical species with any significant
stability) that has the highest free energy
during a reaction.
The combination of a substrate and an enzyme
creates a new reaction pathway whose
transition state energy is lower than that of the
reaction in the absence of enzyme.
Decrease the activation energy
Initial velocity
Enzyme activity is commonly expressed by the initial velocity
( V0) of the reaction being catalyzed.
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The reaction rate is defined as the product
formation per unit time.
The slope of product concentration ([P])
against the time in a graphic representation is
called initial velocity.
It is of rectangular hyperbolic shape.
Reaction velocity curve

Vi
rectangular hyperbola
First order reaction:
The rate of the reaction is
directly proportional to [S] only when [S] is low.
Zero-order reaction:
When [S] is sufficiently high,
the velocity approach maximum velocity (Vmax).
Michaelis-Menten Equation

The mathematical expression of the product
formation with respect to the experimental
parameters

Michaelis-Menten equation describes the
relationship between the reaction rate and
substrate concentration [S]
Effect of substrate
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At early times in the reaction, the
concentration of the product ([P]) is
negligible and the overall reaction can be
written as (no reverse reaction)
k1
k3
E + S  ES  E + P
k2
Michaelis-Menten Equation
(1913)
v=
Vmax [S]
Km + [S]
Km： Michaelis constant
K 2 + K3
Km=
K1
V m [S]
V=
K m + [S]
•当 [S] << Km时,
Vm
[S]
V=
Km
•当 [S] >> Km时,
V ≌ Vm
Understanding Km

Km is a constant derived from rate constants K = k 1 + k 2
m
k1

Km is, under true Michaelis-Menten conditions, an estimate of
the dissociation constant of E from S, because
k1
k1[ E]S  = k 1[ ES ]
at equilibrium,
E+S
ES
k-1
Kd =
[ E ]S  k 1
=
[ ES ]
k1
Reversible reaction, dissociation constant is
So
small Km means tight substrate binding;
high Km means weak substrate binding.
 Km equals to the substrate concentration at which v=1/2vmax
 Km is characteristic constant of an enzyme-catalyzed reaction.
 Km has units of molarity.
 Km indicates the affinity of E and S.
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Km is independent of [E]. It is determined by
the structure of E, the substrate and
environmental conditions ( ph, T, ionic
strength,)
Km is a characteristic constant of E.
The value of Km quantifies the affinity of the
enzyme and the substrate.
The larger the Km, the smaller the affinity.
Understanding Vmax
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The theoretical maximal velocity
Vmax is a constant
Vmax is the theoretical maximal rate of the reaction - but it
is NEVER achieved in reality
To reach Vmax would require that ALL enzyme molecules
are tightly bound with substrate
Vmax is asymptotically approached as substrate is
increased
The reaction velocity of an enzymatic reaction when the
binding sites of E are saturated with substrates.
It is proportional to [E].
The dual nature of the Michaelis-Menten
equation
Combination of 0-order and 1st-order kinetics
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When S is low, the equation for rate is 1st
order in S
When S is high, the equation for rate is 0-order
in S
The Michaelis-Menten equation describes a
rectangular hyperbolic dependence of v on S!
The turnover number
A measure of catalytic activity
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kcat, the turnover number, is the number of substrate
molecules converted to product per enzyme
molecule per unit of time, when E is saturated with
substrate.
If the M-M model fits, k2 = kcat = Vmax/Et
Values of kcat range from less than 1/sec to many
millions per sec
It can be used to compare catalytic activity of
enzymes.
Enzyme Units are used to define the activity
of an enzyme
IU (international unit) ----International Commission on
Enzymes

One IU of enzyme: the amount that catalyzes the
formation of 1 micromole of product in 1 minute.

One katal is that amount of enzyme catalyzing the
conversion of 1 mole of substrate to product in 1
second.

1 katal equals 6 ×107 IU

Specific activity: enzyme units per mg protein

(Units/mg)
Linear Plots can be derived from the
Michaelis-Menten Equation
v =
Vmax[S]
1
Km+ [S]
v
=
Km+ [S]
Vmax[S]
=
Km
Vmax
1
1
+
[S]
Vmax
Lineweaver-Burk double-reciprocal plot
To determine Km and Vmax
To identify the reversible
repression
y = mx + b
Factors affecting enzyme-catalyzed
reaction
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Substrate concentration
Enzyme concentration
Temperature
pH
Inhibitors
activators
Effect of enzyme

[E] affects the rate of
enzyme-catalyzed
reactions

[S] is held constant.

When [S]>>[E], V=[E]
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Optimal T :
T at which an enzyme has
the maximal catalytic power.
35℃-40℃ for warm blood
species
.
Reaction rates increase by
2 folds for every 10℃ rise.
Higher T will denature the
enzyme.
It is not a characteristic
constant of an enzyme
Bell-shaped curve
Enzyme activity
Effect of T
optimum
temperature
Temperature ( °C )
Effect of pH
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Optimal pH
at which the enzyme has
the maximal catalytic
power.
pH 7.0 is suitable for
most enzymes
Particular examples:
Ph (pepsin)= 1.8
Ph (trypsin)=7.8
inhibitors

Inhibitors are certain molecules that can
decrease the catalytic rate of an enzymecatalyzed reaction.

Inhibitors can be normal body metabolites
and foreign substances (drugs and toxins)
Enzyme Inhibition

Enzyme can be inhibited by inhibitors. Inhibitors are tools for
scientists to understand enzymes. Inhibitors are also in many
cases pharmaceutical reagents against diseases;

Inhibitors inhibit enzyme function by binding with enzymes. The
binding reaction can be either reversible or irreversible;

Reversible inhibitors associate with enzymes through noncovalent interactions. Reversible inhibitors include three kinds:
 Competitive inhibitors;
 Non-competitive inhibitors;
 Un-competitive inhibitors

Irreversible inhibitors associate with enzymes through covalent
interactions. Thus the consequences of irreversible inhibitors is
to decrease in the concentration of active enzymes.
Irreversible inhibition




Inhibitors are covalently bound to the essential groups of
enzymes.
Inhibitors cannot be removed with simple dialysis or superfiltration.
Binding can cause a partial loss or complete loss of the
enzymatic activity.
3 different types:



Group Specific Reagent:
 - inhibitor does not resemble substrate
Substrate Analogue:
 - inhibitor resembles substrate
Suicide Inhibitors:
 - inhibitor resembles substrate, turns "dangerous" after processed
by enzyme
Example 1
organophosphorous compounds
RO
R’O
RO
O
P
X
+ HO
E
Organophosphorou Acetylcholine
s compound:
esterase
pesticides
R’O
O
P
O
Inhibited AchE
+
HX
E
Acid
Regeneration of active enzyme
——pyridine aldoxime methyliodide (PAM)
E
E
Inhibited AchE
PAM
active AchE
Inactive inhibitor
Example 2 compounds with heavy metal ions
Hg2+ \ Ag+ \ As 3+
lewisite
Inhibited enzyme
-SH-enzyme
Heavy metal containing chemicals bind to the –SH groups to inactivate the enzymes
Regeneration of active enzyme
----2,3- dimercaptopropanol (BAL)
Inhibited enzyme
BAL
Active
enzyme
Compond with As

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

Example 3 Antibiotic penicillin
Penicillin is a suicide inhibitor
Resemble substrate
Binds at enzyme active site
(not an "inhibitor" yet)
Processed by enzyme via
normal catalytic mechanism
Becomes a chemically active
intermediates that modifies
and inactivates the enzyme
irreversibly
Good candidate for drug due
to minimal side effect
Prevent the synthesis of the bacterial cell wall, killing the cell
Reversible inhibition

Inhibitors are bound to enzymes non-covalently

The reversible inhibition is characterized by an
equilibrium between free enzymes and inhibitorbound enzymes.
competitive inhibition
non-competitive inhibition
uncompetitive inhibition
Competitive inhibition

Competitive inhibition - inhibitor (I) binds only to
E, not to ES

Competitive inhibitors share the structural
similarities with that of substrates.

Competitive inhibitors competes for the active sites
with the normal substrates.

Inhibition depends on the affinity of enzymes and
the ratio of [E] to [S]

At high substrate concentration, the effect of a
competitive inhibitor can be overcome.
Competitive Inhibitors
E+S
+
I
k3
k-3
EI
v =
k1
K-1
k2
ES
vmax [ S ]
[I ]
[ S ] + K m (1 +
)
KI
v
E+P
Km increases
vmax unchanged
1/v
vmax
+inhibitor
Slope=Km/vmax
Km
Km(1+[I]/KI)
[S]
-1/Km
1/vmax
-1/(Km(1+[I]/KI))
1/[S]
Vmax is not affected.
Km is increased.
1
vi
1
′
Inhibition feature

As [S] increases, the effect of inhibitors is
reduced, leading no change in Vmax

Due to the competition for the binding sites,
Km rises, equivalent to the reduction of the
affinity.

example






FH4( tetrahydrofolate) is a coenzyme in the nucleic acid synthesis,
and FH2(dihydrofolate) is the precursor of FH4.
Bacteria cannot absorb FH4(folic acid )directly from environment.
Bacteria use p-amino-benzoic acid (PABA), Glu and dihydropterin to
synthesize FH2. but human body cannot
one was infected by bacterium he could taken sulfa drug such as
sulfanilamide
Sulfanilamide derivatives share the structural similarity with PABA,
blocking the FH2 formation as a competitive inhibitor.
Bacteria would take sulfanilamide to synthesize its FH4, but the
product is useless. Finally bacteria would be killed because of lack of
normal FH4. because human body is unable to synthesize FH4, there
is no effect when taking the drug.
Clinical application of competitive inhibition
Competitive inhibitors
Succinate
dehydrogenase
succinate
fumeric acid
malonate oxaloacetic acid malic acid
PABA
p-amio-benzoic acid(PABA)
dihydropterin
glutamic acid
sulfanilamide
Dihydrofolate
synthetase
Dihydrofolate
synthetase
dihydrofolate
tetrahydrofolate
(FH2)
(FH4)
Noncompetitive inhibition

Inhibitors bind to other sites rather
than the active sites on the free
enzymes or the E-S complexes.
The E-I complex formation does
not affect the binding of
substrates.
The E-I-S complexes do not
proceed to form products
Conformational changed
Reducing the [ E-S]
Vmax, reduce, unchanged Km.
The effect of a noncompetitive inhibitor cannot be
overcome at high substrate concentrations.
Noncompetitive Inhibitors
E+S
+
k1
k-1
ES
+
I
I
KI’
KI
EI + S
EIS
k2
v =
E+P
[S ]

[S ] + K m
vmax
[I ]
(1 +
)
KI
Km unchanged
vmax decreases
v
+inhibitor
1/v
vmax
(1+[I]/KI)/Vmax
Vmax/(1+[I]/KI)
Km
Km
[S]
-1/Km
Slope=Km/vmax
Slope= Km(1+[I]/KI)/vmax
1/vmax
1/[S]
Uncompetitive inhibition





Uncompetitive inhibitors bind only to the enzyme-substrate
complexes.
The E-I-S complexes do not proceed to form products.
The E-I-S complexes do not backward to the substrates
and enzymes.
This inhibition has the effects on reducing both Vmax and
Km
Commonly in the multiple substrate reactions
Uncompetitive Inhibitors
E+S
k1
k-1
ES
+
k2
I
Km decreases
vmax decreases
E+P
v =
KI’
EIS
Slope unchanged
v max
[S ]

Km
K
(1 + I )
[S ] +
K
[I ]
(1 + I )
[I ]
+inhibitor
v
1/v
vmax
(1+ KI/[I])/Vmax
Vmax/(1+KI/[I])
Km/(1+ KI/[I]) Km
[S]
-1/Km
- (1+ KI/[I])/Km
Slope=Km/vmax
Slope= Km/vmax
1/vmax
1/[S]
Summary of Classes of Inhibitors




Competitive inhibition - inhibitor (I) binds only to E, not to
ES
Noncompetitive inhibition - inhibitor (I) binds either to E
and/or to ES
Uncompetitive inhibition - inhibitor (I) binds only to ES,
not to E. This is a hypothetical case that has never been
documented for a real enzyme, but which makes a
useful contrast to competitive inhibition.
Mixed inhibition-when the dissociation constants of (I) to
E and ES are different. The inhibition is mixed.
Inhibitor
Type
Competitive
Inhibitor
Noncompetitive
Inhibitor
Uncompetitive
Inhibitor
Binding Site on Enzyme
Specifically at the catalytic site, where it
competes with substrate for binding in a
dynamic equilibrium-like process. Inhibition
is reversible by substrate.
Binds E or ES complex other than at the
catalytic site. Substrate binding unaltered, but
ESI complex cannot form products. Inhibition
cannot be reversed by substrate.
Binds only to ES complexes at locations other
than the catalytic site. Substrate binding
modifies enzyme structure, making inhibitorbinding site available. Inhibition cannot be
reversed by substrate.
Kinetic effect
Vmax unchanged
Km increased.
Km unaltered
Vmax decreased
Vmax decreased
Km decreased.
activators
Activator: substances enable non-active enzyme to
become active one or low-active enzyme to become highactive one.
Metals such as Mg2+, K+, Mn2+, etc.
essential activator: enable non-active enzyme to
become active one . For example, Mg2+ for hexokinase.
non-essential activator : enable low-active enzyme to
become high-active one . For example, Cl- for salivary amylase.
Sample Questions

What is the v/Vmax ratio when [S]=5Km?
[S ]vmax
v =
[S ] + K m


5K m 5
[S ]
=
=
=
vmax [S ] + K m 5K m+ K m 6
v
Draw a Lineweaver-Burk plot if Vmax=100 mmol/mL,
Km=2mM.
Draw the new lineweaver-Burk plot on the same plot as
above if a competitive inhibitor is added. [I]=0.5 mM,
KI=1mM.
Enzyme 3
regulation of enzyme
Enzymes need to be active in the right
place at the right time
Inappropriate expression can lead to uncontrolled
growth or cell (and organism) death

Many biological processes take place at a specific
time; at a specific location and at a specific speed.

The catalytic capacity is the product of the enzyme
concentration and their intrinsic catalytic efficiency

The key step of this process is to regulate either
the enzymatic activity or the enzyme quantity.
Reasons for regulation

Maintenance of an ordered state in a timely
fashion and without wasting resources

Conservation of energy to consume just
enough nutrients

Rapid adjustment in response to
environmental changes.
How do cells control specific
biochemical reactions?





Control of enzyme activity
Control of enzyme levels
Control of enzyme location
Control of substrate availability
Removal or conversion of reaction
products
What Factors Influence Enzymatic Activity?
The number of enzyme molecules
Enzyme Regulation
The activity of enzyme molecules

Genetic regulation of enzyme synthesis and decay determines
the amount of enzyme present at any moment.

The availability of substrates and cofactors usually determines
how fast the reaction goes.

As product accumulates, the apparent rate of the enzymatic
reaction will decrease.

Enzyme activity can be regulated allosterically.

Enzyme activity can be regulated through covalent modification.

Modulator proteins regulate enzymes through reversible binding

Regulation of enzyme activity also can be accomplished in
other ways.
The thousands of enzyme-catalyzed chemical
reactions in living cells are organized into a series of
biochemical or metabolic pathways. Metabolic and
other processes are controlled by altering the
quantity or the catalytic efficiency of key enzymes.
Enzyme
activity
regulation




Enzyme
amount
regulation


allosteric regulation
covalent modification
Zymogen and activation of
zymogen
isoenzymes
induction and repression
(genetic control)
enzyme degradation
Regulation of enzyme activity




allosteric regulation
covalent modification
Zymogen and activation of zymogen
isoenzymes
Allosteric regulation





Allosteric enzymes are those whose activity can be
adjusted by reversible, non-covalent binding of a specific
modulator to the regulatory sites, specific sites on the
surface of enzymes.
Allosteric enzymes are normally composed of multiple
subunits which can be either identical or different.
The multiple subunits are
Catalytic subunits: Where active sites are located.

Substrate binds here
Regulatory subunits: Effectors bind here
Allosteric regulation: Metabolites binds to
region of outside the active site, and change the
conformation, and thus the enzyme activity.
Allosteric regulators do not bind to
the active site of the enzyme
Activation or inhibition of an enzyme’s activity due to binding
of an activator or inhibitor at a site that is distinct from the
active site of the enzyme.
Allosteric enzymes: some definitions
1. Allosteric = “other site” other than active site
2. Regulatory molecules called, effectors, modulators,
regulatory molecules
3. Homotropic regulation: regulation by substrate at
active site
4. Heterotropic regulation: regulation by molecule
NOT substrate ( end products), at allosteric site
5. Few enzymes are allosteric
6. Allosteric enzymes DO NOT exhibit M-M kinetics
kinetic plot of V versus [S] is sigmoidal shape.
Allosteric regulation can be positive or negative
Properties of regulatory enzymes
1. Their kinetics do not obey
the Michaelis-Menten
equation.
In the jargon of allostery,
substrate binding is
cooperative.
S-shaped / hyperbolic curve
2. The regulatory effect by
product is allosteric
inhibition.
3. Regulatory or allosteric enzymes are regulated by activation.
4. Allosteric enzymes have an oligomeric organization.
5. The regulatory effects are achieved by conformational changes
occurring in the protein when effector metabolites bind..
Allosteric regulators shift the substrate
dependence curve
In the above plot, the allosteric activator decreases the Km of the
enzyme, while the allosteric inhibitor increases the Km of the
enzyme
Allosteric activators stabilize the
high affinity state of the enzyme
Allosteric inhibitors stabilize the
low affinity state of the enzyme
Physiology of allosteric enzymes
Consider biochemical pathways:
-Homotropic regulation, substrate activation
activation
E1
A
E2
B
E3
E4
C
D
E5
E
F
-Heterotropic regulation, end product inhibition
E1
A
E2
B
E3
C
E4
D
E5
E
F
inhibition
A
B
D
E
G
H
C
F inhibits C->D
F partially inhibits
A -> B
I
Feedback inhibition
Feedback inhibition, i.e., building up of
a pathway’s end product ultimately
slows the entire pathway, is often
realized through allosteric enzymes.
The enzyme catalyzing the first step of
a synthetic pathway is often an
allosteric enzyme.
Concerted Model

Assumes 2 conformation states: R
&T

Binding of substrate induces all
subunits to change to R state.

No T-R hybrids.

Allows for + cooperativity only.
+ cooperativity
T state
R state
Sequential Model



The binding of substrate switches conformation of only
the subunit to which it is bound.
Conformational change in one subunit may  or  the
affinity of other subunits have for the substrate.
Allows for + or - cooperativity.
Sequential Model
T
T
R
T
R
R
R
R
R
R
T
T
T
T
T
T
T
R
R
R
Covalent modification
Covalent modification: Some groups of an
enzyme can bind to certain chemical groups by
covalent bond and change the enzyme activity.




A variety of chemical groups on enzymes could be
modified in a reversible and covalent manner.
Such modification can lead to the changes of the
enzymatic activity.
The activity of many enzymes are regulated by
reversible covalent modifications.
Phosphorylation/dephosphorylation, the most
common reversible covalent modification, is a
highly effective means of switching the activity
of target enzymes.
Common modification






Phosphorylation- dephosphorylation
adenylation - deadenylation
methylation - demethylation
uridylation
- deuridylation
ribosylation - deribosylation
acetylation - deacetylation
The most common modification is the addition and removal of
a phosphate group: phosphorylation and dephosphorylation
respectively.
Protein kinases catalyze the transfer of a phosphate
group from an ATP molecule to the side chains of
Ser, Thr, or Tyr residues in proteins.
Protein phosphatases catalyze the hydrolysis of
phosphoryl groups attached to proteins, thus
reversing the effects of kinases.
Protein kinases

Phosphorylation introduces a bulky group bearing two
negative charges, causing conformational changes that
alter the target protein’s function
Pi
Pi
Classification of protein kinases
Class I: Ser/Thr protein kinases,
PKA, PKC, MAPK, CaM-PhK
Class II: Ser/Thr/Tyr protein kinases
Class III: Tyr protein kinases
ATP, manganese ion
Features of covalent modification






Two active forms (high and low)
Covalent modification
Energy needed
Amplification cascade
Some enzymes can be controlled by
allosteric and covalent modification.
Reversible, usually requires one enzyme for
activation and another for inactivation
zymogen
zymogen: Some enzymes are synthesized
as larger inactive precursors called
proenzymes or zymogens.
Zymogen activation: Zymogens are
activated by the irreversible hydrolysis of
one or more peptide bonds under certain
conditions.
The process of zymogen activation is actually the
process of active site formation.
Mechanism of zymogen activation
zymogen
Definite condition
Selective proteolysis
Conformational change
Formation active center
Examples include the hormone insulin (proinsulin),
pepsinogen, trypsinogen, etc.
Regulation of digestive enzymes
B-peptide A-peptide C-peptide
cut
C-peptide
proinsulin
insulin
Pepsinogen is converted to pepsin by
autocatalytic proteolysis at pH 2
pepsinogen
(inactive)
Secretion into
stomach (pH - 2)
autocatalytic
cleavage of
pepsinogen after
amino acid 44
pepsin
(active)
Enzymes involved in protein digestion, blood clotting, and
tissue and bone remodeling are synthesized in an inactive
conformation, then activated by proteolytic cleavage
Zymogen
Pepsinogen
Chymotrypsinogen
Trypsinogen
Procarboxypeptidase
Proelastase
Prothrombin
Fibrinogen
Factor VII
Factor X
Proinsulin
Procollagen
Procollagenase
Active Enzyme
Function
Pepsin
protein digestion
Chymotrypsin
protein digestion
Trypsin
protein digestion
Carboxypeptidase protein digestion
Elastase
protein digestion
Thrombin
blood clot formation
Fibrin
blood clot formation
Factor VIIa
blood clot formation
Factor Xa
blood clot formation
Insulin
plasma glucose homeostasis
Collagen
component of skin and bone
Collagenase
remodeling processes during
metamorphosis, etc.
Physiological significance of zymogen
activation
 Prevent autodigestion of the secretory organ itself.
For example, premature activation of pancreatic
zymogens ( trypsinogen, chymotrypsinogen and
proelastase) leads to the condition of acute pancreatitis
 Keep a rapid and amplified response to condition
changes because zymogens are storage of enzymes.
Isozymes
Isoenzymes: Multiple forms of an enzyme
which catalyze the same reaction, but differ
from each other in their amino sequence,
physicochemical properties and immuno
characters.
 Isoenzymes maybe encoded by different genes or
alleles.
 Isoenzymes may also come from translation of different
mRNAs which are transcripted from the same gene.
What is an isozyme?
(1) Isozymes are physically distinct forms of the
same enzyme.
(2) Isozymes may differ from each other by
differences in their amino acid sequences or by
the presence of different posttranslational
modifications in each isozyme.
(3) The relative abundance of different isozymes
varies for different tissues. The ability to control
which isozymes are expressed in a particular cell
allows each cell to adjust the enzyme activity
based on the specific conditions that exist in the
cell.
Isoenzymes
Lactate dehydrogenase (LDH)
LDH is a tetramer of two non-identical subunits
H subunit
Tetramer LDH (M.W. 130,000)


M subunit
Subunit: H (Chr12)
M (Chr11)
Each monomer can be either heart or muscle type
Five different isozymes of lactate dehydrogenase
exist: H4, H3M, H2M2, HM3, and M4
Tissue distribution specificity
Isozymes are enzymes with slightly different subunits
Quaternary forms of Lactate Dehydrogenase (LDH)
LDH
All of the lactate dehydrogenase isozymes catalyze the
interconversion between lactate and pyruvate
O-
O
C
HO
NAD +
NADH
+ H+
O
C
O-
CH
C
CH3
CH3
L-lactate
NAD +
NADH
+ H+
O
pyruvate
Lactate dehydrogenase
(Muscle type)
O-
O
C
HO
NAD +
NADH
+ H+
O
C
O-
CH
C
CH3
CH3
L-lactate
NAD +
NADH
+ H+
Lactate dehydrogenase
(Heart type)
O
pyruvate
Each isozyme of lactate dehydrogenase can
be separated by chromatography
+
-


Different tissues have
different levels of each of
the lactate dehydrogenase
isozymes
During myocardial
infarction, endothelial
cells rupture, releasing
their contents into the
bloodstream. This
increases the serum levels
for lactate dehydrogenase
isozymes 1 and 2 and can
be used as a diagnostic
tool.

For each lane, the five different isozymes of lactate
dehydrogenase are number 1-5 (with the isozyme
containing all heart type monomers being 1 and all muscle
type monomers being 5).

The lane labeled “HEART” is from the serum of a patient
with a myocardial infarction (heart attack),

the lane labeled “NORMAL” is normal serum,
and the lane labeled “LIVER” is from a patient with liver
disease.

Diagnostic Enzyme Analysis
Normal serum
Myocardial
infarction
Acute hepatitis
Regulation of enzyme quantity
（1）synthesis
induction
repression
（2）degradation
 Cells can synthesize specific enzymes in response to
changing metabolic needs, a process referred to as
enzyme induction.
 The synthesis of certain enzymes may also be
specifically inhibited, a process referred to as enzyme
repression.
Drug design: Transition state analogues

Enzymes have evolved to recognize the
transition state of the reaction they
catalyze

To design an enzyme inhibitor, we should
try to mimic the transition state of the
reaction, not the substrates or products
Clinical applications





Fundamental concepts:
Plasma specific or plasma functional enzymes:
normally present in the plasma and have specific
functions.
High activities in plasma than in the tissues.
Synthesized in liver and enter the circulation.
Impairment in liver function or genetic disorder
leads to a fall in the activities.
Non-plasma specific or plasma non-functional
enzymes: either totally absent or at a low
concentration in plasma.


In the normal turnover of cells, intracellular
enzymes are released into blood stream.
An organ damaged by diseases may elevate
those enzymes.
Diagnostic applications






Usefulness:
-enzyme assays provide important information
concerning the presence and severity of diseases.
-provide a means of monitoring the patient’s
response
Approaches;
-measuring the enzymatic activities directly
-used as agents to monitor the presence of
substrates.
Therapeutic applications






Successful therapeutic uses
- streptokinase: treating myocardial infarction;
preventing the heart damage once
administrated immediately after heart attack
-asparaginase: tumor regression
Several limits
-can be rapidly inactivated or digested
-may provoke allergic effects
summary

Enzymes produced by living cells are specific
biocatalysts which have high specificity and
efficiency in catalytic reactions.

Simple enzymes and conjugated enzymes
cofactors:
Coenzymes
prosthetic groups

Active site of enzyme. Essential groups
Enzyme kinetics studies on the factors which
affect the velocity of enzyme reaction.
 They
includes T, pH, [S], [E], activator, and
the inhibitors.
Michaelis-Menten
equation.
v=
Vmax [S]
Km + [S]

Lineweaver-Burk double-reciprocal plot

Km equals to the substrate concentration at
V0 =Vmax/2.
In competitive inhibition reactions, the apparent Km is increased, while Vmax
is not affected.
In noncompetitive inhibition reaction, Km is not affected, while Vmax is
lowered.
In uncompetitive inhibition reaction, Km and Vmax are all lowered.
+inhibitor
1/v
(1+[I]/KI)/Vmax
-1/Km
1/v
Slope=Km/vmax
Slope=Km/vmax
Slope= Km(1+[I]/KI)/vmax
1/vmax
1/[S]
-1/Km
-1/Km
1/vmax
1/[S]
-1/(Km(1+[I]/KI))
+inhibitor
1/v
(1+ KI/[I])/Vmax
+inhibitor
Slope=Km/vmax
Slope= Km/vmax
1/vmax
1/[S]
Enzyme regulation:
Allosteric regulation; Zymogen; Isozyme;
Covalent modification;
Exploring the old and
deducing the new makes a
teacher.
Thank you!