Chapter 6: Outline-1
Properties of Enzymes
Classification of Enzymes
Enzyme Kinetics
Michaelis-Menten Kinetics
Lineweaver-Burke Plots
Enzyme Inhibition
Catalysis
Catalytic Mechanisms
Cofactors
6P1-1
Chapter 6: Outline-2
Catalysis cont.
Temperature and pH
Detailed Mechanisms
Genetic Control
Enzyme Regulation
Covalent Modification
Allosteric Regulation
Compartmentation
6P1-2
Introduction
The proteins which serve as enzymes,
Mother Nature’s catalysts, are globular
in nature. Because of their complex
molecular structures, they often have
exquisite specificity for their substrate
molecule and can speed up a reaction
by a factor of millions relative to an
uncatalyzed reaction. This
presentation will describe how
enzymes function.
6P1-3
6.1 Properties of Enzymes
A catalyst enhances the rate of reaction
but is not permanently altered.
Catalysts work by decreasing the
activation energy for a reaction.
The structure of the active site of the
enzyme (shape and charge
distribution) is used to optimally orient
the substrate for reaction.
The energy of the enzyme-substrate
complex is then closer to the TS.
6P1-4
Activation Energy, Eact
Transition state
Activation
energy, DG
Free
Reactants
Energy
Free energy change
(DG) for the reaction
Products
Reaction progress
An enzyme speeds a reaction by
lowering the activation energy. It does
this by changing the reaction pathway.
6P1-5
Activation Energy-2
An enzyme lowers the activation energy
but it does not change the standard
free energy change (DG) for the
reaction nor the Keq.
A catalyst cannot make an endergonic
reaction exergonic or vice versa.
Most enzymes are temperature/pH
sensitive and will not work outside
their normal temperature/pH range
because the enzyme is denatured.
6P1-6
Enzymes Models
In the lock-and-key model, the enzyme is
assumed to be the lock and the
substrate the key. The two are made to
fit exactly. This model fails to take into
account the fact that proteins can and
do change their conformations to
accommodate a substrate molecule.
6P1-7
Enzymes Models-2
The induced-fit model of enzyme action
assumes that the enzyme conformation
changes to accommodate the substrate
molecule. Eg.
Conformation
changes
Enzyme and
substrate do
not “fit”.
6P1-8
6.2 Classification of Enzymes
The International Union of Biochemistry
(IUB) classifies and names enzymes
according to the type of chemical
reaction it catalyzes.
Enzymes are assigned a four-number
class and a systematic two-part name.
A shorter recommended name is also
suggested.
Alcohol dehydrogenase is:
alcohol:NAD+ oxidoreductase
(E.C. 1.1.1.1)
6P1-9
Enzyme Classes
1. Oxidoreductases catalyze redox
reactions. Eg. Reductases or
peroxidases
2. Transferases transfer a group from
one molecule to another. Eg.
Transaminases, transcarboxylases
3. Hydrolases cleave bonds by adding
water. Eg. Phosphatases or
peptidases
6P1-10
Enzyme Classes-2
4. Lyases catalyze removal of groups to
form double bonds or the reverse. Eg.
decarboxylasaes or synthases
5. Isomerases catalyze intramolecular
rearrangements. Eg. epimerases or
mutases
6. Ligases bond two molecules together.
Many are called synthetases. Eg.
carboxylases
6P1-11
Enzyme Classes-3: Examples
Class Example
1
alc dehydrogenase
Reaction
O
CH3CH2OH + NAD+
CH3CH + NADH + H+
glucose + ATP 
glucose-6-phosphate
+ADP
2
hexokinase
3
chymotrypsin polypeptide + H2O 
peptides
6P1-12
Enzyme Classes-3: Examples
Class Example
Reaction
4
pyruvate
OO
CH3C C O + H+
decarboxylase
O
CH3CH + CO2
5
alanine
racemase
L-alanine
6
pyruvate
carboxylase
D-alanine
OO
CH3C C O + HCO3_
ATP
ADP+Pi
O
OO
O C CH2C C O
6P1-13
6.3 Enzyme Kinetics
Kinetics is the field of chemistry that
studies the rate and mechanism of a
reaction.
Rates are usually measured in terms of
how many moles of reactant or product
are changed per time period.
A mechanism is a detailed step-by-step
description of how a reaction occurs at
the molecular level.
6P1-14
The Rate Equation-1
A  P
Init. Rate = vo = - D[A] or D[P]
Dt
Dt
D = change in, [A] = molarity and t is
time.
Disappearance of reactants is negative
so the quantity has a negative sign to
make all rates positive.
First order: Rate = D[A] = k[A]1
Dt
6P1-15
The Rate Equation-2
x
Rate = k [A] The rate equals the
experimentally determined rate
constant, k, times the concentrations
of A to some experimentally
determined power, x. Values for x are
frequently 0, 1 or 2.
ALL RATE EQUATIONS ARE
DETERMINED EXPERIMENTALLY!!
6P1-16
The Rate Equation-3
A + B  P
Init. Rate = vo = - D[A] or -D[B] or D[P]
Dt
Dt
Dt
Rate = -k[A]1 [B]1 (overall second order)
If B is water (in large excess) then the
reaction appears to be first order in A
and is said to be pseudo first order.
Rate’ = k’[A]1 [B]0 = k’[A]1
6P1-17
The Rate Equation-4
A + A  P
Second order: Rate = D[A] = k[A]2
Dt
Two molecules of A collide to give P.
A  P
Zero order: Rate = D[A] = k[A]0
Dt
Concentration of A has no effect on rate.
6P1-18
Rate Equation Example
For the reaction Gly-Gly + H2O  2 Gly
Use the data to determine x and y in the
rate equation: Rate = k[di-G]x[H2O]y
[di-Gly]
a) 0.1
b) 0.2
c) 0.1
[H2O]
0.1
0.1
0.2
Rate, Ms-1 x 10-2
1
2
2
Water constant and di-Gly doubled (a+b).
Rate doubles. X=1
Di-Gly constant and water doubles (a+c).
Rate doubles. Y=1
6P1-19
Enzyme Rxns: Type 1
Chymotrypsin cleaves proteins at the
COOH end of aromatic side chain AAs.
At low substrate concentrations, the
reaction is first order in substrate.
As the concentration of substrate
increases, the order changes and
approaches zero.
A graph of velocity vs substrate conc. is
hyperbolic. (See graph, #22)
6P1-20
Enzyme Rxns: Type 1
Chymotrypsin:
Hyperbolic
plot
Zero order
Conc. At ½ max velocity
First order
6P1-21
Enzyme Rxns: Type 2
Aspartate transcarbamoylase (ATCase)
catalyzes the reacton between
aspartate and carbamoyl phosphate.
This reaction leads ultimately to the
synthesis of nucleobases needed for
DNA and RNA synthesis.
Velocity as a function of aspartate
concentration gives a sigmoidal plot.
(See #24)
6P1-22
Enzyme Rxns: Type 2
ATCase:
Sigmoidal plot
6P1-23
Enzyme Rxns-cont.
The plots for cymotrypsin and aspartate
transcarbamoylase should remind us
of the oxygen binding curves for
myoglobin and hemoglobin
respectively.
Thusly, chymotrypsin is a nonallosteric
and ATCase is an allosteric enzyme.
We need two different models to explain
these enzymes behaviors.
6P1-24
Michaelis-Menten Kinetics-1
M-M kinetics explains the behavior of
nonallosteric enzymes. It assumes an
enzyme-substrate complex is formed.
E+S
k1
k2
E-S
k3
E+P
At low substrate concentrations, the reaction
is first order with respect to substrate.
At high substrate concentrations, the enzyme
is saturated with substrate. The order is
zero and a Vmax occurs.
6P1-25
Michaelis-Menten Kinetics-2
At low P concentrations (initial rate)
Assumed: k2 negligible vs k1
Rate of formation of ES
equals rate of degradation.
Rate = DP/Dt = k3[ES]
k1[E][S] = (k2 + k3)[ES] (Steady State)
[ES] = [E][S]
(k2 + k3)/k1
Km = (k2 + k3)/k1
Michaelis constant
6P1-26
Michaelis-Menten Kinetics-3
Michaelis and Menten also derived what
is now known as the Michaelis-Menten
equation.
Vmax = max velocity
Vmax[S]
The
lower
the
K
,
the
m
v=
[S] + Km
greater the affinity for
complex formation.
An enzymes’s kinetic properties can be
used to determine its catalytic
efficiency. (Next slide.)
6P1-27
Michaelis-Menten Kinetics-4
kcat = Vmax/[Et] = turnover number
kcat = molecules of S converted to
product per unit time with enzyme
saturated
[Et] = total enzyme concentration
V= (kcat / Km) [E][S]
(kcat / Km) is a rate constant where
[S]<< Km and the constant reflects the
combined effect of binding and
catalysis.
6P1-28
Michaelis-Menten Kinetics-5
Turnover numbers for some enzymes
follow. They vary greatly!!
Enzyme
kcat (s-1)
Catalase
10,000,000
Chymotrypsin
190
Lysozyme
0.5
Note: catalyse turns over 10 milliion
molecules of substrate per sec!!
6P1-29
Michaelis-Menten Kinetics-6
Substrate concentration at ½ Vmax is
termed the KM (Michaelis constant) for
the reaction.
KM is difficult
to measure by
this method as
Vmax must be
estimated.
A linear plot
gives better
results.
6P1-30
Lineweaver-Burk Plot
A Lineweaver-Burk plot for nonallosteric
enzymes gives a straight line and
better data to determine KM.
1 KM 1 1
+
=
V Vmax [S] Vmax
In the form y = mx + b:
1/V is y (V is the measured velocity (rate) of
the reaction), 1/[S] is x, KM/Vmax is the slope
and 1/Vmax is the y intercept.
6P1-31
Lineweaver-Burk Plot
A L-B plot of 1/V vs 1/[S] is shown below
slope is
KM
Vmax
1
V
x intercept is
-1
KM
0
y intercept is
1
Vmax
1/[S]
6P1-32
Enzyme Inhibition
Inhibitors interfere with enzyme action.
They may be reversible or irreversible.
The three kinds of reversible inhibitors
are competitive uncompetitive and
noncompetitive .
A competitive inhibitor looks structurally
like the substrate and binds to the
enzyme at the active site.
An uncompetative inhibitor binds only to
the enzyme-substrate complex.
6P1-33
Enzyme Inhibition-2
A noncompetitive inhibitor does not look
like substrate and binds at a site other
than the active site.
6P1-34
Competitive Inhibitor
E+S
+
I
K1
K2
E-S
K3
E+P
K12 K11
EI + S
NR
6P1-35
Competitive Inhibitor-2
Since a competitive inhibitor competes
with substrate for the active site, its
influence can be negated with large
concentrations of substrate. Thus the
Vmax remains constant.
Since the velocity is slower compared to
normal substrate concentrations, the
slope of the L-B line increases and the
KM increases.
The effect of a competitive inhibitor on a
L-B plot is shown on slide 42.
6P1-36
Uncompetitive Inhibitor
E+S
K1
K2
E-S
+
I
K3
E+P
K12 K11
EIS
6P1-37
Uncompetitive Inhibitor-2
Since an uncompetitive inhibitor binds
only to the enzyme-substrate complex,
adding more substrate will increase the
rate but not to the original values
without inhibitor.
Commonly observer when the enzyme
binds to more than one substrate.
6P1-38
Noncompetitive Inhibitor
E+S
+
I
EI + S
K1
K2
E-S
+
I
ES
K3
E+P
NR
6P1-39
Noncompetitive Inhibitor-2
For a noncompetitive inhibitor, the
velocity of the reaction is slowed at all
substrate concentrations. Thus the
Vmax is permanently lowered.
The slope of the L-B line increases but
KM stays constant.
The effect of a noncompetitive inhibitor
on a L-B plot is shown on slide 42.
6P1-40
Kinetics: Inhibition
Competitive:
Vmax same
KM changes
1
V
Noncompetitive:
Vmax changes
KM same
0
No inhibitor
1/[S]
6P1-41
The kinetic behavior of allosteric
enzymes, catalysis in general, and
specific enzyme mechanisms are
discussed in the next slide series:
Enzymes Part 2
6P1-42