BT201 Module 5

Enzymes

Tanmoy Sarkar

Tezpur University

April 24, 2024

Protein Functions

Enzyme Nomenclature

Enzymes are classified by the reaction they catalyse:

Protein Functions

Chemical Kinetics
Elementary reactions

A −→ P

A reaction may take place through a sequence of elementary

reactions:

A −→ I1 −→ I2 −→ P
Protein Functions

Chemical Kinetics
Rate equations

aA + bB + · · · + zZ −→ P

Rate equation
The rate of a process is proportional to the frequency at which the
reacting molecules come together, that is the products of the
concentrations of reactants

Rate = k [A]a [B]b . . . [Z ]z (1)

Protein Functions

Chemical Kinetics
Rate equations

A few nomenclatures
k = a proportionality constant, known as the rate constant,
order of the reaction = (a + b + . . . + z) = sum of the
exponents of the rate equation

A −→ P, is a first-order reaction,
2A −→ P, is a second-order reaction
Protein Functions

Chemical Kinetics
Rates of reactions

The instantaneous rate of a reaction (v) can be assessed by

measuring [A] or [P] as a function of time:

d [A] d [P]
v =− = (2)
dt dt
For first-order reaction, A −→ P:
d [A]
v =− = k [A] (3a)
dt
For second-order reaction, A + B −→ P:
d [A] d [B]
v =− =− = k [A] [B] (3b)
dt dt
Protein Functions

Chemical Kinetics
First order rate equation

For first-order reaction, A −→ P, rearranging Equation 3a gives:

d [A]
= d ln [A] = −k · dt (4)
[A]

Integrating Equation 4, from [A0 ] (initial concentration of A) to

[A] (concentration of A at time t) gives:
Z [A] Z t
d ln [A] = −k dt
[A]0 0

This results in the first order rate equation:

ln [A] = ln [A]0 − kt (5)

Protein Functions

Chemical Kinetics
First order rate equation

Equation 5 produces a linear equation with t and ln[A] as variables:

Graphical representation of Equation 5 and determination of rate constant (k)

Protein Functions

Chemical Kinetics
Transition state

A spontaneous bimolecular
reaction involving 3 atoms
(A, B, C):

A − B + C −→ A + B − C

The reaction continues by

forming a high-energy
(unstable) transition
complex:
A transition state diagram of a spontaneous reaction
A...B ...C
Protein Functions

Chemical Kinetics
Transition state

Concentration of the transition state is small ⇒ formation of

transition state is the rate-limiting step
Larger the ∆Gintermediate (free energy difference between the
transition state and the reactants) ⇒ less stable the transition state
⇒ slower the rate of the reaction
Rate of the reaction:
depends on concentration of reactants
decreases exponentially with ∆Gintermediate
∆Gintermediate is also called the free energy of activation or the
activation barrier
In multistep reactions, the slowest reaction acts as a bottleneck, and
is known as the rate-limiting step
Catalysts act by lowering the activation barrier
activation barrier lowered for both forward and reverse
reactions
equilibrium constant for the reaction remains unchanged
Protein Functions

Enzyme Kinetics
Michaelis-Menten Equation

A simple enzyme-catalyzed reaction (E is enzyme, S is substrate,

ES is enzyme-substrate complex, P is product):

k12 k
E + S ⇌ ES −→ E +P
k−1

The general rate of this reaction is:

d [P]
v= = k2 [ES] (6)
dt
The overall rate of production of ES is:

d [ES]
= k1 [E ] [S] − k− 1 [ES] − k2 [ES] (7)
dt
Protein Functions

Enzyme Kinetics
Michaelis-Menten Equation

A few assumptions of enzyme-catalyzed

reactions:
1 Assumption of equilibrium
k−1 >> k2 , so the formation of the
ES complex reaches equilibrium:
k−1 [E ] [S]
KD = = (8)
k1 [ES]
KD is the dissociation constant of the ES complex

2 Assumption of steady-state
After the initial transient phase,
the [ES] remains constant, till
substrate is nearly exhausted:
Progression curves of a simple Michaelis-Menten
d [ES] reaction. Note, the change in [ES] and [E] in the
=0 (9) shaded block are essentially zero, hence are in
dt steady-state. The transient phase precedes the
shaded steady-state.
Protein Functions

Enzyme Kinetics
Michaelis-Menten Equation

Direct measurement of [E] and [ES] are not feasible, but total
enzyme concentration ([E]T ) is:

[E ]T = [E ] + [ES] (10)

Substituting Equations 10 & 9 into Equation 7 gives:

k1 ([E ]T − [ES]) [S] = k−1 + k2 [ES] (11)

Equation 11 upon rearranging, becomes:

[ES] k−1 + k2 + k1 [S] = k1 [ET ] [S] (12)
Protein Functions

Enzyme Kinetics
Michaelis-Menten Equation

Dividing both sides of Equation 12 by k1 and solving for [ES] gives:

[E ]T [S]
[ES] = (13)
KM + [S]

KM is called the Michaelis-Menten constant and is defined as:

k−1 + k2
KM = (14)
k1
Substituting Equation 13 into Equation 6, we get the initial rate
of the reaction (v0 ):
!
d [P] k2 [E ]T [S]
v0 = = k2 [ES] = (15)
dt KM + [S]
t=t s
ts = time when steady-state is first achieved
Protein Functions

Enzyme Kinetics
Michaelis-Menten Equation

Maximal velocity of a reaction

(Vmax ) occurs at high substrate
concentrations when the enzyme
is totally saturated and is
entirely in ES complex form:

Vmax = k2 [E ]T (16)

Combining Equations 15 & 16,

we get the Michaelis-Menten
equation: A plot of initial rate of reaction (v0 ) with substrate
concentration ([S]) of a simple Michaelis-Menten
reaction.
N.B.: when [S] = KM , then v0 = Vmax /2
Vmax [S]
v0 = (17)
KM + [S]
Protein Functions

Enzyme Kinetics
Lineweaver-Burke plot

A better method for determining

Vmax and KM was formulated by
Hans Lineweaver and Dean Burk,
by taking the reciprocals of
Equation 17:
!
1 KM 1 1
= + (18)
v0 Vmax [S] Vmax

This is a linear equation in 1/v0 A double-reciprocal graph. Note the large effect of small
errors at small [S] (large 1/[S]), and the crowding at
& 1/[S], and the plot is called large [S]

Lineweaver-Burke plot or
double-reciprocal graph
Protein Functions

Enzyme Kinetics
Inhibition

A general model for competitive inhibition:

2k1 k
E + S ⇌ ES −→ E +P
k−1

+
I
↿⇂ KI
EI + S −→ NO REACTION

In competitive inhibition, inhibitor binds reversibly with enzyme,

and is in rapid equilibrium:
[E ] [I ]
KI = (19)
[EI ]
Protein Functions

Enzyme Kinetics
Inhibition

The EI complex is catalytically inactive ⇒ A competitive

inhibitor acts by reducing the amount of free enzyme available for
binding to substrate.
Taking EI into account, Equation 10 becomes:

[E ]T = [E ] + [EI ] + [ES] (20)

[E] can be expressed in terms of [ES] by rearranging Equation 7:

KM [ES]
[E ] = (21)
[S]
[EI] can be expressed by rearranging Equation 19:
[E ] [I ]
[EI ] = (22)
KI
Protein Functions

Enzyme Kinetics
Inhibition

Taking Equations 21 & 22 and substituting into Equation 20 gives:

(   )
KM [I ]
[E ]T = [ES] 1+ +1 (23)
[S] KI

Rearranging and solving Equation 23 for [ES] gives:

[E ] [S]
[ES] =  T  (24)
[I ]
KM 1 + K I
+ [S]

Thus, initial rate of reaction (v0 ) becomes:

k [E ]T [S]
v0 = k2 [ES] = 2  (25)
[I ]
KM 1 + KI + [S]
Protein Functions

Enzyme Kinetics
Inhibition

Thus, for competitive

inhibition:
 
[I ]
α= 1+ (26)
KI

and, substituting for Vmax

from Equation 16 gives:

Vmax [S]
v0 = (27)
αKM + [S] Michaelis-Menten plot of competitive inhibition
Protein Functions

Enzyme Kinetics
Inhibition

Taking reciprocal of
Equation 27 gives:
!
1 αKM 1 1
= +
v0 Vmax [S] Vmax
(28)
The obtained
double-reciprocal plot is
linear,
with a slope of
αKM /Vmax ,
a y-intercept (1/v0 ) of
1/Vmax ,
a x-intercept (1/[S]) of Double-reciprocal plot of competitive inhibition
-1/αKM
Protein Functions

References

Biochemistry by Voet & Voet

Lehninger’s Principles of Biochemistry by Nelson & Cox