Lecture 25

12/01/11
Figure 11-29
Chymotrypsin stabilizes the tetrahedral
oxyanion transition states
Amide nitrogens of Ser195 and Gly193 form
an oxyanion hole in which the substrate
carbonyl oxygen is hydrogen-bonded to the
amide N-H groups.

Figure 11-30a
Figure 11-30b
Forming Tetrahedral transition state increases the interaction of the carbonyl
oxygen with the amide N-H groups by:
1. Conversion of the carbonyl double bond to the longer tetrahedral
single bond brings the oxygen atom closer to the amide hydrogens
2. Hydrogen bonds between the charged oxygen and the amide
hydrogens are significantly stronger than the hydrogen bonds with the uncharged
carbonyl oxygen.
HIV Protease Inhibitors
Protease Inhibitors has extended life of HIV patient
Inhibit HIV protease together with a reverse transcriptase inhibitor like
AZT can reduce the human immunodeficiency virus (HIV) to
undetectable levels in about 40 to 50% of infected individuals.
Four of the protease inhibitors approved for use in humans by the U.S.
Food and Drug Administration are : Crixivan (r) by Merck, Invirase (r) by
Hoffman- LaRoche , Norvir (r) by Abbott, and Viracept (r) by Agouron.
These drugs were all developed from a structure-based design strategy;
that is, the drug molecules were designed to bind tightly to the active site
of the HIV-1 protease.
Backbone of OH-group in all these substances inserts between the two
active-site carboxyl groups of the protease.
Chapter 12
Enzyme Kinetics
Enzyme kinetics
Kinetics is the study of reaction rates (t dependent
phenomena)
Rates of reactions are affected by
Enzymes/catalysts
Substrates
Effectors
Temperature
How do enzymes reduce activation
energy (G)?

1. By lowering the free energy of the transition
state (), e.g., by binding the transition state
tightly
2. By changing the reaction pathway by which
reactants react to form products
Each reaction step has its own transition state
with its own activation energy (G).
The overall rate of the catalyzed reaction is
dictated by the slowest step in a multistep
reaction.
BINDING = the essence of enzyme action
binding of SUBSTRATE to form an ES COMPLEX
binding of TRANSITION STATE more tightly than the
substrate
Binding occurs at ACTIVE SITE of enzyme.
Subsequent chemical events can then occur.
Active site:
relatively small part of whole enzyme structure
3-dimensional cleft with participating components from
different parts
of primary structure
water often excluded so substrates and intermediates are in
non-aqueous environment (unless H
2
O is a reactant)
Binding uses multiple weak interactions:
1. hydrogen bonds
2. salt links
3. van der Waals interactions
4. hydrophobic effect
Why study enzyme kinetics (reaction rates)?
measurement of velocity = reaction rate
WANT to determine the maximum reaction velocity
that the enzyme can attain and its binding affinities for
substrates and inhibitors
compare activity of same enzyme with different
substrates (understand specificity)
measure amount or concentration of one enzyme in a
mixture by its activity
measure enzyme purity (specific activity = amount of
activity/amount of protein)
study/distinguish different types of inhibitors
development of specific drugs (enzyme
inhibitors)
Chemical kinetics is the study of the
rates of chemical reactions
A P
To simplify it, we look at this one-step conversion
More likely occurs through a sequence of
elementary reactions, each of which is a simple
molecular process, as in
A I J P
I and J represent intermediates

All the elementary reactions required to define the
overall reaction mechanism for A P

Each step of this
reaction is an
elementary step.
Each elementary
step has reactant(s),
a transition state,
and product(s).
Products that are
consumed in
subsequent
elementary reaction
are called
intermediates.
Let us assume that A P is an elementary reaction:
spontaneous and essentially irreversible
Irreversibility is easily assumed if the rate of P conversion to A is
very slow or the concentration of P (expressed as [P]) is negligible
under the conditions chosen.
The velocity, v, or rate, of the reaction A P is the amount of P
formed or the amount of A consumed per unit time, t. That is,




The mathematical relationship between reaction rate and
concentration of reactant(s) is the rate law:



rate constant k has the units of (time)
-1
(sec
-1
)
For an elementary reaction, the order for any reactant is
given by its exponent in the rate equation.

15
Reaction Rates: To measure a reaction
rate we monitor the disappearance of
reactants or appearance of products.
e.g., 2NO
2
+ F
2
2NO
2
F

16
Use experimental data to determine the
reaction order.
If a plot of [A] vs t is a straight line, then the reaction is zero order. Rate is independent of the
concentration of reactant. Doubling the concentration has no effect on the rate.
If a plot of ln[A] vs t is a straight line, then the reaction is 1
st
order. Rate is directly related to the
concentration of the reactant; double reactant increase rate by a factor of 2.
If a plot of 1/ [A] vs t is a straight line, then the reaction is 2
nd
order. Rate is related to the square
of the concentration of the reactant. Doubling the reactant =increase rate by a factor of 4.


First Order Reaction
-6
-5
-4
-3
-2
-1
0
0.0E+00 2.0E+04 4.0E+04 6.0E+04
Time (in seconds)
l
n

[
A
}

(
i
n

m
o
l

/


L
Second Order Reaction
0
50
100
150
200
250
0 500 1000
Time (in seconds)
1
/
[
A
]

(
L

/
m
o
l
)
mA -----> P rate = k[A]
m

Half-Lives
The time required for the initial concentration of
a reactant to be reduced to one-half its initial
value
For a zero order reaction A products; rate = k
t

= [A
o
] / 2k
For a first order reaction A products, rate= k[A]
t

= 0.693 / k
For a second order reaction 2A products or
A + B products (when [A] = [B]), rate = k[A]
2

t

= 1 / k [A
o
]
Determining Half-Lives
To determine a half life, t

, the time required

for the initial concentration of a reactant to be
reduced to one-half its initial value, we need
to know:
The order of the reaction or enough
information to determine it.
The rate constant, k, for the reaction or
enough information to determine it.
Sometimes need to know the initial
concentration, [A
o
]


We calculate the average rate
of a reaction over a time
interval by dividing the change
in concentration over that
time period by the time
interval.
For the change in
concentration of a reactant,
the equation, where the
brackets
mean "concentration of", is


An instantaneous rate is the rate at some instant in
time.
An instantaneous rate is a differential rate:
-A[reactant]/ A t or A[product]/ A t.

We determine an instantaneous rate at time t:
by calculating the negative of the slope of the curve of
concentration of a reactant versus time at time t or by
calculating the slope of the curve of concentration of a
product versus time at time t.

Molecularity
The number of molecules that must
simultaneously interact is defined as the
molecularity of the reaction.
Elementary reactions can be unimolecular
(one molecule involved in given elementary
reaction)
Bimolecular (two molecules collide with each
other in a given elementary reaction), etc.).
Unimolecular Reaction:
A P
Biomolecular Reactions
Consider the more complex reaction, where two molecules must react to yield
products:
A + B P + Q
Assuming this reaction is an elementary reaction, its molecularity is 2; that is, it is a
bimolecular reaction. The velocity of this reaction can be determined from the rate of
disappearance of either A or B, or the rate of appearance of P or Q:
The rate law is: v = k[A][B]
The rate is proportional to the concentrations of both A and B. Because it
is proportional to the product of two concentration terms, the reaction is
second-order overall, first-order with respect to A and first-
order with respect to B.
In a first-order chemical reaction, the conversion of A to P occurs because, at any given
instant, a fraction of the A molecules has the energy necessary to achieve the transition
state.
The rate of any chemical reaction is proportional to the concentration of reactant
molecules (A in this case) having this transition-state energy.
Specifically, AG

is the energy required to raise the average energy of one mole of reactant
(at a given temperature) to the transition-state energy.
The relationship between activation energy and the rate constant of the reaction, k, is
given by the Arrhenius equation:
k = Ae
-

AG/RT
A is a constant for a particular reaction
If the energy of activation decreases, the reaction rate increases
free energy of
activation, AG

Derivation of Enzyme Kinetics
Equations
Start with a mechanistic model
Identify constraints and assumptions
Do the algebra ...

Simplest Mechanism: E + S

ES E + P

- One reactant
- One intermediate
- One product

Michaelis-Menten Kinetics
Lenore Michaelis and Maud L. Menten proposed a general theory of enzyme action in 1913
consistent with observed enzyme kinetics.
The enzyme, E, and its substrate, S, associate reversibly to form an enzyme-substrate
complex, ES:
This association/dissociation is assumed to be a rapid equilibrium, and Ks is the enzyme :
substrate dissociation constant. At equilibrium, rate
reverse
= rate
forward

k
-1
[ES] = k
1
[E][S]
and
The enzyme binds non covalently
to the substrate
the non-covalent ES complex is known as the
Michaelis complex.
Michaelis complexes are stabilized by
molecular interactions (non-covalent).
Michaelis complexes form quickly and
dissociate quickly
The Michaelis-Menten model: equation relating
reaction velocity to substrate concentration for a
system where a substrate S binds reversibly to an
enzyme E to form an enzyme-substrate complex
ES, which then reacts irreversibly to generate a
product P and to regenerate the free enzyme E.
This system can be represented schematically as
follows:

The Michaelis-Menten equation for this system is:

Steady-State Assumption
In 1925 by Briggs and Haldane assume that the
concentration of the enzyme-substrate complex ES
quickly reaches a constant value
ES is formed as rapidly from E + S as it disappears by its
two possible fates:
1. dissociation to regenerate E + S
2. Form E + P.
This assumption is termed the steady-state assumption
and is expressed as
That is, the change in concentration of ES
with time, t, is 0.
Initial Velocity Assumption
The total amount of enzyme is fixed and is given by the formula
Total enzyme, [E
T
] = [E] + [ES]
the rate of [ES] formation is
v
f
= k
1
([ET] - [ES])[S]
where [ET] - [ES] = [E]
The rate of [ES] disappearance is
v
d
= k
-1
[ES] + k
2
[ES] = (k
-1
+ k
2
)[ES]
At steady state, d[ES]/dt = 0, and therefore, v
f
= v
d
.
So, K
1
([ET] - [ES])[S] = (k
-1
+ k
2
)[ES]
Rearranging gives

Michaelis Constant, Km
The ratio of constants (k
-1
+ k
2
)/k
1
is itself a constant
and is defined as the Michaelis constant , K
m


Recall that


Rearranging to

Rate of product formation: v = k
2
[ES]
What does k
2
[ET] mean?
At saturation, [ES] complex is equal to the total
enzyme concentration, [ET] because E=ET-ES
(enzyme is saturated), therefore E=0

The initial velocity v then equals k
2
[E
T
] = V
max
.
Written symbolically, when [S] >> [E
T
] (and K
m
),
[E
T
] = [ES] and v = V
max
V
max
= k
2
[E
T
]
Substituting this relationship into the expression
for v gives the Michaelis- Menten equation

This equation says that the rate of an enzyme-
catalyzed reaction, v, at any moment is
determined by two constants, K
m
and V
max
, and
the concentration of substrate at that moment.
When [S] = K
m
, v = V
max
/2

That is, K
m
is defined by the substrate
concentration that gives a velocity equal to one-
half the maximal velocity.
E + S

ES E + P
The enzyme is either free ([E]) or bound ([ES]):
[E
o
]= [ES] + [E]
At high [S] all of the enzyme is tied up as ES (i.e., [E
o
] [ES], according
to Le Chatelier's Principle)
At high [S] the enzyme working at full capacity (v
max
).
The full capacity velocity is determined by the catalytic constant, k
cat
.
k
cat
= turnover #: number of moles of substrate produced per unit of time
per enzyme active site.


k
cat


velocity = v =
d[P]
dt
= k
cat
[ES]
v
max
= k
cat
[E
0
]
k
cat
=
v
max
[E
0
]
[E
o
] =[ET]
E + S

ES E + P
For any enzyme it is possible (pretty easy) to
determine k
cat
.

To understand and compare enzymes we need to
know how well the enzyme binds to S (i.e, what
happens in the first part of the reaction.) k
cat
does not
tell us anything about how the enzyme binds to the
substrate.
so, (turn the page and learn about K
D
and K
M
).

k
cat

The initial velocity approaches a
maximum at high [S].
The initial velocity increases with [S] at
low [substrate].

Initial velocity
The initial velocity increases with [S].

Michaelis-Menten Kinetics
1. Enzyme (E) and substrate (S) reversibly and
quickly form a non-covalent ES complex.
2. This first step (binding) is followed by some kind
of chemical transformation, and dissociation to
give product (P) and enzyme (E).
Michaelis-Menten Kinetics: Assumptions
1. k
1
,k
-1
>>k
2
(i.e., the first step is fast and is always at
equilibrium).

2. d*ES+/dt 0 (i.e., the system is at steady state.)









3. There is a single reaction/dissociation step (i.e., k
2
=k
cat
).
4. S
Tot
= *S+ + *ES+ *S+
5. There is no back reaction of P to ES (i.e. *P+ 0). This
assumption allows us to ignore k
-2
. We measure initial
velocities, when *P+ 0.


d[ES]
dt
= rate of formationof ES
rateof breakdownof ES
~ 0 (at steady state)
The time dependence of everything (in a
Michaelis-Menten Reaction)
steady-state approximation
Since V
max
is the reaction velocity at
saturating substrate concentration, it is
equal to k
cat
[ES] when [ES] = [E
T
].
K
M
is the substrate concentration required to reach
half-maximal velocity (v
max
/2).
Michaelis-Menten Parameters:
Significance of K
M
K
M
is the apparent dissociation constant of the ES complex. A
dissociation constant (K
D
) is the reciprocal of the equilibrium
constant (K
D
=K
A
-1
). K
M
is a measure of a substrates affinity for the
enzyme (but it is the reciprocal of the affinity).

If k
1
,k
-1
>>k2, the K
M
=K
D
.

K
M
is the substrate concentration required to reach half-maximal
velocity (v
max
/2). A small K
M
means the sustrate binds tightly to the
enzyme and saturates (maxs out) the enzyme.

the microscopic meaning of K
m
depends on the details of the
mechanism.


Michaelis-Menten Parameters:
Significance of k
cat
k
cat
: For the simplest possible mechanism, where ES is the only
intermediate, and dissociation is fast, then k
cat
=k
2
, the first order
rate constant for the catalytic step.
If dissociation is slow then the dissociation rate constant also
contributes to k
cat
.
If there are multiple catalytic steps (see trypsin) then each of those
rate constants contributes to k
cat
.
If one catalytic step is much slower than all the others (and than the
dissociation step), than the rate constant for that step is
approximately equal to to k
cat
.
k
cat
is the turnover number: indicates the rate at which the enzyme
turns over, i.e., how many substrate molecules one catalytic site
converts to substrate per second.
The microscopic meaning of k
cat
depends on the details of the
mechanism].
Michaelis-Menten Parameters:
Significance of k
cat
/K
M
k
cat
/K
M
is the catalytic efficiency.

k
cat
/K
M
is an apparent second order rate constant

v=k
cat
/K
M
[E]
0
[S]

k
cat
/K
M
can be used to estimate the reaction velocity from the
total enzyme concentration ([E]
0
)

k
cat
/K
M
is the specificity constant. It is used to distinguish and
describe various substrates.


Data analysis
It would be useful to have a linear plot of
the MM equation
Lineweaver and Burk (1934) proposed the
following: take the reciprocal of both sides
and rearrange.
Collect data at a fixed [E]
0
.