Enzyme Kinetics

-fructofuranosidase:

Sucrose + H2O

glucose + fructose

When [S] [E] : the rate is zero order with respect to sucrose.
Initial rate no longer increases at S higher than S4

The Michaelis-Menten Equation

ET = E + ES

v= Vmax = k2 ET

v/Vmax = k2 ES / k2 ET

ES/ ET

v/Vmax = ES/ ET
v/Vmax = ES/ E + ES How to know ES?

This equation cannot be explicitly integrated, however, without simplifying assumptions,

two possibilities are:

1. Assumption of equilibrium. Leonor Michaelis and Maud Menten, building on the

work of Victor Henri, assumed that k-1 k2, so that the first step of the reaction reaches
equilibrium.

ES = E * S / Ks
Ks is the dissociation constant of the first step in the enzymatic reaction

The Michaelis-Menten Equation

1.

Assumption of steady-state. Figure illustrates the progress curves of the various

participants in reaction

under the physiologically common conditions that substrate is in great excess over
Enzyme ([S] [E]).

ES maintains a steady state and [ES] can be

treated as having a constant value:

The so called steady state assumption, a more

general condition than that of equilibrium, was
first proposed in 1925 by G. E. Briggs and B. S.
Haldane

The Michaelis-Menten Equation

Solving for [ES]:

ES = E * S /(k-1+k2)/k1

The Michaelis constant, KM ,

is defined as

Therefore:

ES = E * S / KM

The Michaelis-Menten Equation

The expression of the initial velocity (v0) of the reaction, the velocity
at t=0, thereby becomes

v/Vmax = ES/ (E + ES)

v/Vmax = (E*S)/Km/ (E + (E*S)/Km )
v/Vmax = S/ Km / (1 + S/Km)

v/Vmax = S / Km + S
This expression, the Michaelis-Menten
equation, is the basic equation of
enzyme kinetic.

The maximal velocity of a reaction, Vmax occurs at high substrate concentrations when
the enzyme is saturated, that is, S>> Km, and ET is entirely in the ES form v= Vmax
when

Significance of the Michaelis Constant

The Michaelis constant, KM, has a simple operational definition. At the substrate
concentration at which [S] = KM, this equation
yields v0 = Vmax/2 so that
KM is the substrate concentration at which the reaction velocity is half maximal

Significance of the Michaelis Constant

The magnitude of KM varies widely with the identity of the enzyme and the nature of the substrate.
It is also a function of temperature and pH. The Michaelis constant can be expressed as

Since Ks is the dissociation constant of the Michaelis complex, as Ks decreases, the enzymes affinity
for substrate increases. KM in therefore also a measure of the affinity of the enzyme for its substrate,
provided k2/k1 is small compared to Ks, that is k2 k-1 so that the ES
P reaction proceeds more
slowly than ES reverts to E + S

kcat/KM Is a Measure of Catalytic Efficiency

We can define the catalytic constant, kcat, of an enzyme as

This quantity is also known as the turnover number of an enzyme because it is the number of
reaction processes (turnovers) that each active site catalyzes per unit time.

Turn Over Numbers of Enzymes

kcat (s-1)

Enzymes

Substrate

Catalase

H2O2

Carbonic anhydrase

HCO3-

400,000

Acetylcholinesterase

Acetylcholine

140,000

b-Lactamase

Benzylpenicillin

Fumarase

Fumarate

RecA protein (ATPase)

ATP

40,000,000

2,000

800
0.4

The number of product transformed from substrate

by one enzyme molecule in one second
Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.263

kcat/KM Is a Measure of Catalytic Efficiency

When [S] KM, very little ES is formed. Consequently, [E] [E]T, so

reduces to a second-order rate equation:

The quantity kcat/KM is a measure of an enzymes catalytic efficiency.

There is an upper limit to the value of kcat/KM : It can be not greater than k1; that is, the decomposition of ES
to E + P can occur no more frequently than E and S come together to form ES. The most efficient enzymes
have kcat/KM values near to the diffusion-controlled limit of 108 to 109 M-1.s-1

Chymotrypsin Has Distinct kcat /Km to Different Substrates

=

O
R O
H3CCNCCOCH3
H H

kcat / Km

R=
Glycine

1.3 10-1

Norvaline

CH2CH2CH3

3.6 102

Norleucine

CH2CH2CH2CH3

3.0 103

Phenylalanine CH2

1.0 105

(M-1 s-1)
Adapted from Mathews et al (2000) Biochemistry (3e) p.379

Lineweaver-Burk or double-reciprocal plot

Analysis of Kinetic Data

S >> Km
vi=Vmax
Vmax= k2Et

v= Vmax = k2 ET
Vmax= 10 x 10 -6 M/seg Km=10 x10-5 M
Si en el ensayo se usaron 5mg/L de preparacin enzimtica, entonces:

k2= 10 x 10 -6 /5 = 2 x 10 -6 moles/mg seg

Qu predicciones podemos hacer a partir de esta informacin?

Al iniciar:
t = 0, S = So
A cualquier tiempo:
T=t S=S
X = (So-S)/So

- dS/dt = vi = So dX/dt

 As is the case with most reactions, an increase in

temperature will result in an increase in kcat for an
enzymatic reaction.
From general principles, it can be determined that the
rate of any reaction will typically double for every
10C increase in temperature.
Many enzymes display maximum temperatures around
40C, which is relatively close to body temperature.
There are enzymes that are isolated from thermophilic
organisms that display maxima around 100C, and
some that are isolated from psychrophilic organisms
that display maxima around 10C.

1/v = 1/ Vmax + Km/Vmax (1/S) + (1 / Ki Vmax) S

S pequeas

1/v = 1/ Vmax + Km/Vmax (1/S) + (1 / Ki Vmax) S

S grandes

1/v = 1/ Vmax + Km/Vmax (1/S) + (1 / Ki Vmax) S

Enzyme Inhibition
Many substances alter the activity of an enzyme by reversibly combining with it in a way
what influence the binding of substrate and/or its turnover number. Substances that reduce
an enzymes activity in this way are known as inhibitors

Competitive Inhibition
A substance that competes directly with a normal substrate for an enzymes substratebinding site is known as a competitive inhibitor.

Here it is assumed that I, the inhibitor, bind

reversibly to the enzyme and is in a rapid
equilibrium with it so that
And EI, the enzyme-inhibitor complex, is
catalytically inactive. A competitive inhibitor
therefore reduces the concentration of free
enzyme available for substrate binding.

Enzyme Inhibition
Competitive Inhibition

This
is
the
Michaelis-Menten
equation that has been modified by a
factor, , which is defined as

Is a function of the inhibitors concentration and

its affinity for the enzyme. It cannot be less than 1.

Enzyme Inhibition
Competitive Inhibition
Recasting

in the double-reciprocal form yields

A plot of this equation is linear and has a slope of KM/Vmax, a 1/[S] intercept
of -1/ KM, and a 1/v0 intercept of 1/ Vmax

Enzyme Inhibition
Uncompetitive Inhibition
In uncompetitive inhibition, the inhibitor binds directly to the enzyme-substrate complex
but not to the free enzyme

In this case, the inhibitor binding step has the dissociation constant

The uncompetitive inhibitor, which need not resemble the substrate, presumably distorts
the active site, thereby rendering the enzyme catalytically inactive.

Enzyme Inhibition
Uncompetitive Inhibition
The double-reciprocal plot consists of a family of parallel lines with slope KM/Vmax, 1/v0
intercepts of /Vmax and 1/[S] intercept of -/KM

Enzyme Inhibition
Mixed Inhibition (noncompetitive inhibition)

A mixed inhibitor binds to enzyme sites that participate in both substrate binding and
catalysis. The two dissociation constants for inhibitor binding

Double-reciprocal
plots
consist of lines that have
the slope KM/Vmax, with a
1/v0 intercept of /Vmax
and 1/[S] intercept of -/
KM

Two-Stage Catalysis of Chymotrypsin

O Nitrophenol acetate
CH3CO
NO2

Acylation

O
CH3C

NO2

O
C

O-H
C

+ H2O

Nitrophenol

CH3COOH

Deacylation (slow step)

Kinetics of reaction

OC

HO

Two-phase
reaction

Time (sec)
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.169

DESVIACIONES A M&M

Ecuaciones de balance de todas

las formas enzimticas
dE/dt = -k1 E*S + k-1 ES + k3 ES

De la suposicin de estado estacionario:

dE/dt = 0
-k1 E*S + k-1 ES + k3 ES

dES/dt = k1 E*S ES (k-1 + k2)

dES/dt = 0
ES = k1 E*S / (k-1 + k2)

dES/dt = k2 ES k3 ES

dES/dt = 0
ES = k2 ES / k3

v = dP1/dt = k2 ES
v = dP2/dt = k3 ES
v = k3 k2 ES / k3

v = k2 k1 E*S / (k-1 + k2)

v = dP2/dt = k3 ES
Vmax = k3 ET

ET= E + ES + ES

Vmax = k3 (E + ES + ES)
Vmax = k3 (E + k1 E*S / (k-1 + k2) + k2 ES / k3)
Vmax = k3 (E + k1 E*S / (k-1 + k2) + k2 k1 E*S / (k-1 + k2) / k3 )

Vmax = k3 E + k1 k3 E*S / (k-1 + k2) + k2 k1 E*S / (k-1 + k2)

v / Vmax

=
k2 k1 E*S / (k-1 + k2)
k3 E + k1 k3 E*S / (k-1 + k2) + k2 k1 E*S / (k-1 + k2)

v / Vmax

v / Vmax

v / Vmax
Vmax = k3 ET

k2 k1 S
k3 (k-1 + k2) + k1 k3 S + k2 k1 S
k2 k1 S
k3 (k-1 + k2) + k1 S (k2 + k3)

k2 S / (k2 + k3)
k3 (k-1 + k2) / k1(k2 + k3) + S

k2 k3 ET S / (k2 + k3)
k3 (k-1 + k2) / k1 (k2 + k3) + S

Dividir entre:
k1 (k2 + k3)

K-2+

Vmax f = k2 k3 ET
k-2 + k3 +k2
Vmax r = k-1 k-2 ET
k-1 + k2 +k-2
Ks = k-1 k-2 + k-1 k3 + k2 k3
k1( k2 + k-2 + k3)

Kp

= k-1 k-2 + k-1 k3 + k2 k3

k-3( k-1 + k2 + k-2)

K-2+
Keq = Vmax f * Kp
Vmax r * Ks

v = Vmax f Kp S Vmax r Ks P
KsKp + KpS + KsP

v = Vmax f S P / Keq
Ks + S + (Ks/Kp) P