Download Chapter 3. Enzymes

Chapter 3. Enzymes
1. Introduction
An enzyme is a protein catalyst that speeds up a
reaction without being changed itself.
1) Characteristics of enzyme catalytic reactions:
a) enzyme catalytic reactions are thermodynamically
possible. Enzymes accelerate reactions by factors
of at least a million, but not change the equilibrium;
b) enzymes are highly specific for their reactants
which are so called “substrates”;
c) many enzyme catalytic activities are
regulated;
d) enzymes possess all physicochemical
properties of protein and hence are affected
by temperature, pH, organic solvents, or
other conditions which denature proteins.
2) Composition of enzyme molecules:
Enzymes
Simple enzymes: consist
of only protein
Conjugated enzymes: consist
of protein & nonprotein constituent
For a conjugated enzyme,
Holoenzyme = apoenzyme + cofactor
Cofactor: small organic or inorganic molecule
that an enzyme requires for its activity.
Cofactors is divided into two types:
Coenzymes: small organic molecules, usually
derived from vitamins. They loosely bind to
apoenzymes.
Prosthetic groups: are inorganic ions or organic
molecules which are covalently bound to
apoenzymes.
Some important coenzymes
Coenzymes
Nicotinamide adenine
dinucleotide (NAD+)
Nicotinamide adenine
dinucleotide phosphate (NADP+)
Flavin adenine dinucleotide (FAD)
Flavin mononucleotide (FMN)
Thiamin pyrophosphate (TPP)
Pyridoxal phosphate
Coenzyme A (CoA)
Biocytin
Function
transfer H+, e
Sources
Vit. PP
transfer H+, e
Vit. PP
transfer H
transfer H
transfer -CHO
transfer -NH2
transfer acyl
transfer CO2
Vit. B2
Vit. B2
Vit. B1
Vit. B6
Pantothenic
Biotin
Some enzymes require both organic and
inorganic cofactors, such as cytochrome
oxidase which contains heme and Cu.
Some enzymes consist of more than one
protein subunit (polypeptide chain) with
quaternary structures, such as monomeric,
oligomeric and multimeric enzymes.
3) Active site of enzyme: is the region that
binds the substrate and converts it into
product. The active site must be a 3-d entity
which can be bound by substrate(s) via noncovalent weak forces (e.g. salt bridge, Hbond, hydrophobic interaction, Van De
Waals force).
The active sites are usually clefts or crevices.
Two models for enzyme-substrate binding:
a) Lock-and-key model: the shape of the
substrate fits the active site of the enzyme
just like a key to its lock.
b) Induced-fit model: interaction between the
substrate and the enzyme induces a
conformational change in the active site
such that the active site is complementary to
the substrate.
Models for enzyme-substrate binding
(a) Lock-and-key model, (b) Induced-fit model
4) Essential groups in the active site
There are two types of essential groups in the
active site of enzyme:
a) Binding groups: specifically bind to the
substrate to form enzyme-substrate
complex.
b) catalytic groups: catalyze conversion of
the substrate to product.
5) Substrate specificity of enzymes
The specificity of an enzyme for its substrate
depends on the 3-d structure of the active
site—only the molecule that binds to the
active site to form enzyme-substrate
complex can be converted into product.
e.g. trypsin, chymotrypsin, and elastase have
different structures of active sites which are
specified for their substrates.
Enzyme catalytic specificity can be classified
into three types:
a) Absolute specificity: the enzyme can only
catalyze one reaction of its specific
substrate. e.g.
urease
Urea + H2O
2 NH3 + CO2
b) Relative specificity: the enzyme can
catalyze a reaction of a group of
compounds. e.g.
AA1-AA2…Arg(Lys)-AA(n) -AA (n+1) …
H2O
trypsin
AA1-AA2…Arg(Lys) + AA(n) -AA (n+1) …
c) Stereo specificity: the enzyme can only
catalyze the reaction of one of the isomers
of its specific substrate, such as D or L
isomers, cis or trans isomers. e.g.
L-amino acid oxidase
a-keto acid
L-amino acid
O2
NH3 + H2O2
Induced fit hypothesis: interaction between
substrate and enzyme results in a
conformational change at the active site of
the enzyme, so that the substrate can bind
the enzyme to form a [E-S]complex.
Substrate binding sites of three enzymes
6) Classification of enzymes
7) Isoenzymes
Isoenzymes are different forms of an enzyme
which catalyze the same reaction but exhibit
different physical or kinetic properties.
e.g. several enzymes with different structures
catalyze the same reaction showing
different pH optimum, substrate affinity, or
effect of inhibitors.
For example, lactate dehydrogenase (LDH) has five
isoenzymes: H4, H3M, H2M2, HM3, and M4. They
catalyze the same reaction as following:
LDH
Pyruvate + NADH + H+
lactate
M subunits predominate in muscle and liver whereas
H subunits in the heart.
2. Mechanism of enzymatic catalysis
1) Thermodynamics: G  E-TS
in which G is the free energy change of a
system undergoing a transformation at
constant pressure and temperature;
E=Eb-Ea (energy change of a system
from the start Ea to the end Eb); T is the
temperature; S is a measure of the degree
of randomness or disorder of a system,
called entropy.
If G<0, then the reaction can occur
spontaneously;
If G=0, then the reaction has reached
the equilibrium– no net change can
take place;
If G>0, then the reaction can not occur
spontaneously, but the reaction can
occur when free energy is input.
The G provides no information about
the rate of a reaction.
2) Mechanism of enzymatic catalysis: an
enzyme raises the reaction rate by
stabilizing the transition state of a
chemical reaction and decreasing the
activation energy of the substrate(s).
3. Enzyme kinetics
Enzyme kinetics is the study of the rates of
enzyme-catalyzed reactions.
Enzyme velocity: is the initial rate (V0) of the
reaction being+ catalyzed. The unit of
enzyme velocity is mmol/min, or enzyme
unit (U).
V0 is usually measured before 10% of the
substrate has been converted to product.
V0 changes with time
Enzyme activity: refers to the total units of
enzyme in the sample.
Enzyme specific activity: refers to the
number of enzyme units per mg of protein,
or U/mg
Specific activity is a measure of the purity
of an enzyme.
1) Factors that affect V0
Enzyme velocity is affected by:
a) Substrate concentration: before the enzyme is
saturated by the substrate, increase of
substrate concentration [S] will result in
increase of the initial velocity. However,
after saturation increase of [S] has no effect
on V0.
Therefore, the V0-[S] plot is a hyperbolic curve.
Effect of [S] on V0
b) Enzyme concentration: when the
substrate concentration is saturating,
increase of the enzyme concentration
will lead to increase of V0.
c) Temperature: before the temperature
that denature the enzyme, increase of
the temperature will raise the enzyme
catalytic reaction rate.
Optimum temperature: at which the
enzyme velocity is the maximum value.
d) pH: the pH of the solution affects both
the enzyme molecule and the substrate.
Too low or too high pH may denature
the enzyme.
Optimum pH: the pH at which the
enzyme exhibits its maximal activity.
Effects of T and pH on V0
2) Michaelis-Menten model
In this model the enzyme-catalytic reactions
are proposed as:
k1
E+S
k3
ES
E+P
k2
“ES” is the enzyme-substrate complex
(intermediate).
If v=k3 [ES], Vmax=k3 [ET], km=(k2+k3)/k1,
then the following equation may be established:
[S]
v = Vmax
[S] + km
This is the Michaelis-Menten equation, in
which Vmax is the maximal rate and km is
called Michaelis constant.
The meaning of km is expressed as:
When [S]=Km, then v=Vmax/2.
Therefore, Km is equal to the substrate
concentration at which the reaction rate
is half its maximal value.
The unit of Km is the same as that of [S].
3) The double reciprocal plot
The Vmax and Km can be calculated by the
double reciprocal plot:
1
V0
1
=
Vmax
km 1
.
+
Vmax [S]
4. Enzyme inhibition
Inhibition of enzyme activity can be:
• Irreversible inhibition—the enzyme activity
can not recover even after removal of the
inhibitor from the enzyme.
• Reversible inhibition—the enzyme will
regain its full activity after the inhibitor is
removed from the enzyme.
• Reversible inhibition includes competitive
and noncompetitive inhibition.
1) Irreversible inhibition
Irreversible inhibition refers to the permanent
inactivation of the enzyme due to irreversible
binding of the inhibitor to the enzyme.
Usually, the irreversible inhibitor covalently or
noncovalently binds to the active site of the
enzyme with very slow dissociation rate.
e.g. diisopropylphosphofluoridate (DIPF)
covalently binds the Ser-OH in the active site
of acetylcholinesterase.
2) Reversible competitive inhibition
A competitive inhibitor is structurally similar
to the normal substrate for the enzyme. It
diminishes the enzyme activity by
competing with the substrate to bind to the
active site and thus reducing the proportion
of enzyme molecules bound to the substrate.
Competitive inhibition can be overcome by
increasing the [S].
Competitive inhibition
+
+
E
S
ES
+
E
I
EI
E
P
Km increases while Vmax remains constant.
3) Reversible noncompetitive inhibition
A noncompetitive inhibitor binds reversibly at
a non-active site of the enzyme, causing a
conformational change of the enzyme and
decrease in catalytic activity.
Unlike competitive inhibition, noncompetitive
inhibition can not be overcome by
increasing the [S].
Non-competitive
reactions
E+S
+
I
ES
+
I
EI+S
EIS
E+P
+S
E
－S
+
ES
E
P
+S
EI
－S
ESI
Vmax decreases while Km remains constant.
4) Reversible uncompetitive inhibition
A uncompetitive inhibitor binds only to the
enzyme-substrate complex and not to the
free enzyme. It reduces both the Vmax and
Km.
Uncompetitive reactions
E+S
ES E+P
+
I
ESI
+
E
+
S
ES
ESI
E
P
Plots of uncompetitive reactions
1/V
inhibitor↑
No inhibitor
1/S
Vmax decreases, Km decreases.
5. Regulation of enzyme activity
1) Feedback inhibition: refers to the inhibition
of an enzyme activity of the early reaction by
an end-product of the metabolic pathway.
Sequential feedback inibition—the end-product
of one branch of a pathway inhibits the first
enzyme after the branchpoint but allows the
synthesis of the product of another branch.
Feedback inhibition and sequential
feedback inhibition
2) Allosteric regulation: the binding of a
substrate molecule to one active site affects
the binding of substrate molecules to other
active sites in the enzyme.
Allosteric enzymes are multisubunit proteins
with one or more active sites on each
subunit and are regulated by effector
molecules (may be substrate or nonsubstrate molecules). Its characteristic is
that the V0-[S] plot gives a sigmoidal curve
instead of hyperbolic one.
V0-[S] plot of an allosteric enzyme
Allosteric activator or inhibitor: increases or
decreases the reaction rate catalyzed by the
allosteric enzyme, respectively.
3) Other regulations: include chemical
modification, proteolytic activation, and
regulation of enzyme synthesis and
breakdown.