Enzyme Kinetics - Determination of

properties of enzyme inhibition (Ch 3, 7)

recall kinetics (in first year chemistry and

biochemistry)
in medicinal chemistry, enzyme kinetics is used
especially to determine binding affinities of
substrates and inhibitors to enzymes and the type
of inhibition taking place (eg competitive, non-
competitive)
Important in rational drug design

No 1

Enzyme Kinetics

Kinetics background
A -------> P (if spontaneous and essentially
irreversible)

velocity (rate) v = d[P] or v = -d[A] = k[A]

dt dt rate law

Rate = concentration/time (often Msec-1; rate constant

k (time-1, sec-1)
first-order reaction (unimolecular reaction)

No 2
Kinetics

first-order determination generally

No 3

Kinetics

first-order determination generally

No 4
Kinetics

first-order determination generally

No 5

Bimolecular reactions

e.g. A + B -------> P + Q

velocity (rate) v = -d[A] = -d[B] = d[P] = d[Q]

dt dt dt dt

rate law = k[A][B] (k in concentration-1 x time-1, M-1

sec-1)
second-order reaction
first-order wrt A and first-order wrt B

No 6
Enzyme Kinetics, The Michaelis-Menten
Equation
k1 kcat
E+S ES E+P
k 1
E represents "free" enzyme; S represents a substrate
ES represents the Michaelis "enzyme-substrate
complex
P represents the product
k1/k1 is the ratio of rate constants for formation and
dissociation of ES complexes
kcat is the rate constant for reaction of the ES complex
to give enzyme and product
Assumed irreversible because look at kinetics at early
stage, ie initial velocity
No 7

Enzyme Catalysed Reactions

For an enzymatic reaction of S ----> P (S =

substrate, P = product, enzyme concentration
constant)

Initial velocity from [S] or [P] vs t

and looking at initial slope ([ ]/t)

No 8
Enzyme Catalysed Reactions
at low [S], velocity v [S] (first-order)
as [S] increases, v levels off
at high [S], v becomes virtually independent of [S]
and approaches a maximal velocity = Vmax
at high [S] reaction obeys zero-order kinetics
=> saturation effect, i.e. v can increase no
further even though [S] is increased
Such plots are called substrate saturation curves
at saturation every enzyme molecule in the
reaction mixture has its substrate-binding site
occupied
No 9

Why?

No 10
The Michaelis-Menten Equation
k1 kcat
E+S ES E+P
k 1

Defining Vmax = kcat [Etotal] and

KM (Michaelis constant) = (k1 + kcat)/k1
obtain the Michaelis-Menten equation:
Rate = d[P]/dt = Vmax [S]/(KM + [S])
this equation states that the rate of an enzyme
catalysed reaction, v, at any moment is determined by
two constants, KM and Vmax and the concentration of
the substrate at that moment

No 11

The Michaelis-Menten Equation

d[P]/dt = Vmax [S]/(KM + [S])
generally use initial velocity, vi = (d[P]/dt) t=0
minimises complications such as the effects of reverse
reactions, inhibition of the enzyme by its product(s), and
progressive inactivation of the enzyme
- this is also why the rate of the reverse reaction of P + E --->
ES can be regarded as zero

k1 k cat
E + S ES E + P
k -1
1st order
rapid & reversible slowest, rate-det. step
non-reversible
No 12
kcat

Kcat ES E+P

Kcat is the turnover number of an enzyme

it is a measure of the maximal catalytic activity and is
defined as: the number of substrate molecules
converted into product per enzyme molecule per unit
time when the enzyme is saturated with substrate
also known as molecular activity
at saturating [S], velocity = Vmax = Kcat[Etotal]
=> Kcat = Vmax/[Etotal]
Kcat represents the kinetic efficiency of the enzyme,
the higher the value the better the efficiency
catalase Kcat = 40,000,000 s-1; acetylcholine esterase
14,000 s-1
No 13

Vmax and KM
Vmax, the maximum velocity of an enzymatic reaction, is a
measure of an enzymes catalytic efficiency - dependent on
Etotal (and temperature, pH, ionic strength etc). Higher Vmax
better enzyme.
KM is usually regarded as a measure of the affinity an
enzyme has for its substrate. Measured in concentration, e.g.
mM.
KM measures the concentration of substrate at which half the
active sites of the enzyme are filled. It is dependent on
substrate, pH, temperature, ionic strength.
If an enzyme has a small KM it achieves maximal catalytic
efficiency at low substrate concentration. The lower KM the
better the substrate affinity. Effectively a measure of
substrate binding.
No 14
The Michaelis-Menten Equation - Vmax and Km

No 15

Kcat Vmax and Km

Vmax is the maximum rate of the reaction when the

substrate concentration is very high
Km is the substrate concentration that lets the reaction
occur at 1/2 Vmax
Kcat tells you how quickly the reaction actually occurs.

No 16
Vmax and Km

Vmax and Km can be approximated from the substrate

saturation curve (plot v verses [S])
experimentally when [S] >> Km, then v = Vmax (zero-
order wrt [S])
when [S] < Km, then v ~ (Vmax/Km)[S]
Km and Vmax define the rate of the enzyme-catalysed
reaction provided one substrate (or if more only one
[substrate] varied), ES ---> P is irreversible or the
experiment is limited to observing only initial velocities
where [P] = 0, [S]0 > [Etotal] and [Etotal] is held
constant, pH, temperature, ionic strength, etc held
constant
No 17

The Lineweaver-Burk Double Reciprocal Plot

determination of KM and Vmax Vmax difficult to determine
directly

Reciprocals allow the linear

form:
1 KM + [S]
V = Vmax [S]

KM 1 1
= Vmax [S] + Vmax

(y = mx + b)

No 18
The Eadie Hoftsee Plot NO NEED TO
KNOW A more accurate method
for obtaining reaction
determination of KM and Vmax parameters (data points
are not clustered at high
Vmax [S]):

Vmax[S]
Velocity vi

slope = -KM
v = KM + [S]

also rearranges as
v = Vmax - KM v/[S]
Vmax /KM
More complex enzyme
reactions need computer
vi/[S] fitting of multiple reaction
pathways and statistical
analysis to determine kinetic
parameters
No 19

Enzyme Inhibition Kinetics

inhibition of an enzyme reaction involves the reaction
of a drug with the enzyme to form an ENZYME-DRUG
COMPLEX (more generally, an enzyme-inhibitor or EI
complex), which is unreactive
this decreases the amount of "free" enzyme available
for reaction with the substrate and in turn results in
decreased product formation (enzyme inhibition)
in terms of reaction rate, d[P]/dt, less product is
formed in a given time because [ES] is decreased as
some of the enzyme is diverted to form EI complexes
Rate is decreased
No 20
Enzyme Inhibition Kinetics
Ki
enzymes can be inhibited reversibly, E + I EI
Ki
or irreversibly, E + I EI
reversible inhibition is most common for inhibitors that are
used as pharmacodynamic drugs
irreversible inhibition is more common, and desirable, in
chemotherapeutics
Ki = [E][I]/[EI] Ki = inhibitor constant best way to define
effectiveness of inhibitors. Ki strictly a dissociation constant.
the more strongly bound the drug is to the enzyme, the
smaller the value of Ki and the greater the inhibition of the
enzyme

No 21

Classical Reversible Enzyme Inhibition

Competitive inhibition
Most common type of inhibition substrate and
inhibitor compete for the same binding site (active
site) E + S ES P+E

+ I

Ki

EI + S No reaction

Ki = [E] [I]/[EI]

rate = Vmax[S]/(Km + [S]) = 1 + [I]/Ki

No 22
Classical Reversible Enzyme Inhibition
Competitive inhibition

No 23

Classical Reversible Enzyme Inhibition

Competitive inhibition
is a function of the inhibitors concentration and its
affinity for the enzyme. It can not be less than 1.
as [S] approaches infinity, v0 approaches Vmax for any
value of
a high concentration of the substrate can overcome
the effect of the inhibitor
the presence of I makes [S] appear more dilute than it
really is and KM appear larger - a consequence of
binding of I and S to E being mutually exclusive

No 24
Determination of Competitive Inhibition and Ki
do a Lineweaver-Burk double reciprocal plot with no
inhibitor and varying concentrations of inhibitor

No 25

Determination of Competitive inhibition

defining features
at an increasing [I] the velocity decreases (1/v
increases)
the x intercept decreases as [I] increases
the diagnostic criterion for competitive inhibition is that
Vmax is unaffected by [I], i.e. 1/Vmax is constant

No 26
Determination of Ki for Competitive inhibition
Dixon plot

increasing
[substrate]
1/v minM-1

[Inhibitor]M
0

- Ki
No 27

Classical Reversible Enzyme Inhibition

Pure Non-Competitive inhibition

A less common type of reversible enzyme inhibition

by drugs. It is assumed that the inhibitor has its own
binding site (allosteric site)
E + S ES P+E

+ I + I

Ki Ki'

EI + S ESI No reaction

No 28
Classical Reversible Enzyme Inhibition
Pure Non-Competitive inhibition
determination by Lineweaver-Burk double reciprocal
plot

Pure non-competitive
Ki = Ki

No 29
A/Prof Joanne Jamie, CBMS306/842

Classical Reversible Enzyme Inhibition

Pure Non-Competitive inhibition
Lineweaver-Burk double reciprocal plot
- [I] does not alter KM (x intercept remains the same
with/without I)
- Vmax decreases with I present
for non-competitive enzyme inhibition the inhibitor has
its own binding site on the enzyme and that site exists
not only in the free enzyme, but also in the ES
complex

No 30
Classical Reversible Enzyme Inhibition
Pure Non-Competitive vs Competitive Inhibition
Lineweaver-Burk plot

(E + S + I)
1/d[P]/dt 1/d[P]/dt (E + S + I)

(E + S)
(E + S + I)

(E + S)

-1/[S] 0 1/[S] -1/[S] 0 1/[S]

competitive Non-
competitive
No 31

Classical Reversible Enzyme Inhibition

Pure Non-Competitive inhibition
Dixon plot

No 32
Determination of Ki for Competitive inhibition
Dixon plot

increasing
[substrate]
1/v minM-1

[Inhibitor]M
0

- Ki
No 33

Classical Reversible Enzyme Inhibition

Mixed Non-Competitive inhibition
due to alteration of the substrate active site while
binding to the allosteric site
results in some modification of the Lineweaver-Burk
plot

No 34
Classical Reversible Enzyme Inhibition
Mixed Non-Competitive inhibition
Lineweaver-Burk plot

No 35

Irreversible Enzyme Inhibition

inhibitor in this case generally combines irreversibly to the
enzymes substrate active site by covalent attachment - good
for chemotherapeutic drugs
the kinetic effect is similar to that of non-competitive
reversible inhibitors
usually this type of inhibition can be distinguished from the
non-competitive reversible inhibitors since the reaction of I
with E (and/or ES) is not instantaneous. Instead there is a
time dependent decrease in enzymatic activity as E + I
EI proceeds, and the rate of inactivation can be followed
Also, unlike reversible inhibitions, dilution or dialysis of the
enzyme:inhibitor solution does not dissociate the EI complex
and restore enzyme activity

No 36