Enzyme Kinetics - Determination of

properties of enzyme inhibition (Ch 3)

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

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, noncompetitive)
Important in rational drug design

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

= k[A]
rate law
dt
dt
Rate = concentration/time (often Msec-1; rate constant
k (time-1, sec-1)
first-order reaction (unimolecular reaction)

No 1

Kinetics

No 2

Bimolecular reactions

first-order determination generally

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 3

No 4

Enzyme Catalysed Reactions

Enzyme Kinetics, The Michaelis-Menten

Equation
k1
E+S

kcat
ES

E+P

k1

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

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 5

Enzyme Catalysed Reactions

No 6

The Michaelis-Menten Equation

k1

at low [S], velocity v [S] (first-order)

E+S

kcat
ES

E+P

k1

as [S] increases, v levels off

at high [S], v becomes virtually independent of [S]
and approaches a maximal velocity = Vmax

Defining Vmax = kcat [Etotal] and

at high [S] reaction obeys zero-order kinetics

obtain the Michaelis-Menten equation:

KM (Michaelis constant) = (k1 + kcat)/k1

=> saturation effect, i.e. v can increase no

further even though [S] is increased

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

Such plots are called substrate saturation curves

at saturation every enzyme molecule in the
reaction mixture has its substrate-binding site
occupied
No 7

No 8

kcat

The Michaelis-Menten Equation

Kcat

d[P]/dt = Vmax [S]/(KM + [S])

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

E + S

ES

E+P

Kcat is the turnover number of an enzyme

generally use initial velocity, vi = (d[P]/dt) t=0

k1

ES

k cat

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]

E + P

Kcat represents the kinetic efficiency of the enzyme,

the higher the value the better the efficiency

k -1
1st order
rapid & reversible slowest, rate-det. step
non-reversible

catalase Kcat = 40,000,000 s-1; acetylcholine esterase

14,000 s-1
No 9

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.

No 10

The Michaelis-Menten Equation - Vmax and Km

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 11

No 12

Vmax and Km

The Lineweaver-Burk Double Reciprocal Plot

Vmax and Km can be approximated from the substrate

saturation curve (plot v verses [S])

determination of KM and Vmax

Vmax difficult to determine

directly
Reciprocals allow the linear
form:

experimentally when [S] >> Km, then v = Vmax (zeroorder wrt [S])

1
V =

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

(y =

KM + [S]
Vmax [S]
KM 1
Vmax [S]

1
+ Vmax

mx + b)

No 13

The Eadie Hoftsee Plot

determination of KM and Vmax

Velocity vi

Vmax

slope = -KM

A more accurate method

for obtaining reaction
parameters (data points
are not clustered at high
[S]):

Vmax[S]
KM + [S]

also rearranges as
v = Vmax - KM v/[S]

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)

Vmax /KM

vi/[S]

No 14

More complex enzyme

reactions need computer
fitting of multiple reaction
pathways and statistical
analysis to determine kinetic
parameters
No 15

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 16

Enzyme Inhibition Kinetics

enzymes can be inhibited reversibly, E + I

Classical Reversible Enzyme Inhibition

Ki

EI

Ki
or irreversibly, E + I

Competitive inhibition

EI

reversible inhibition is most common for inhibitors that are

used as pharmacodynamic drugs

Most common type of inhibition substrate and

inhibitor compete for the same binding site (active
site)

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 17

No 18

Classical Reversible Enzyme Inhibition

Classical Reversible Enzyme Inhibition

Competitive inhibition

Competitive inhibition

No 19

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 20

Determination of Competitive Inhibition and Ki

Determination of Competitive inhibition

do a Lineweaver-Burk double reciprocal plot with no

inhibitor and varying concentrations of inhibitor

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 21

No 22

Determination of Ki for Competitive inhibition

Classical Reversible Enzyme Inhibition

Dixon plot

Pure Non-Competitive inhibition

A less common type of reversible enzyme inhibition
by drugs

increasing
[substrate]
1/v minM-1

it is assumed that the inhibitor has its own binding site

(allosteric site)

[Inhibitor]M

- Ki
No 23

No 24

Classical Reversible Enzyme Inhibition

Classical Reversible Enzyme Inhibition

Pure Non-Competitive inhibition

Pure Non-Competitive inhibition

determination by Lineweaver-Burk double reciprocal

plot

Lineweaver-Burk double reciprocal plot

Pure non-competitive
Ki = Ki

- [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 25

A/Prof Joanne Jamie, CBMS306/842

No 26

Classical Reversible Enzyme Inhibition

Classical Reversible Enzyme Inhibition

Pure Non-Competitive vs Competitive Inhibition

Pure Non-Competitive inhibition

Lineweaver-Burk plot

Dixon plot

(E + S + I)

1/d[P]/dt

(E + S + I)

1/d[P]/dt

(E + S)

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

-1/[S]

1/[S]

competitive

-1/[S]

1/[S]

Noncompetitive
No 27

No 28

Determination of Ki for Competitive inhibition

Classical Reversible Enzyme Inhibition

Dixon plot

Mixed Non-Competitive inhibition

due to alteration of the substrate active site while
binding to the allosteric site
increasing
[substrate]

results in some modification of the Lineweaver-Burk

plot

1/v minM-1

[Inhibitor]M

- Ki
No 29

No 30

Irreversible Enzyme Inhibition

Classical Reversible Enzyme Inhibition

inhibitor in this case generally combines irreversibly to the

enzymes substrate active site by covalent attachment - good
for chemotherapeutic drugs

Mixed Non-Competitive inhibition

Lineweaver-Burk plot

the kinetic effect is similar to that of non-competitive 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 31

No 32