Chapter 14.

Enzyme Kinetics
 Chemical kinetics
• Elementary reactions
A → P (Overall stoichiometry)

I1 → I2 (Intermediates)

• Rate equations
aA + bB + … +zZ → P
Rate = k[A]a[B]b…[Z]z
k: rate constant

• The order of the reaction (a+b+…+z): Molecularity of the reaction

– Unimolecular (first order) reactions: A → P
– Bimolecular (second order) reactions: 2A → P or A + B → P
– Termolecular (third order) reactions
 Rates of reactions
A → P (First-order reaction)
d[P] d[A]
v= =- = k[A]
dt dt

2A → P (Second-order reaction)
d[P] d[A]
v= =- = k[A]2
dt dt

A + B → P (Second-order reaction)
d[P] d[A] d[B]
v= =- =- = k[A][B]
dt dt dt
 Rate constant for the first-order reaction

d [A]
v=− = k[A]
dt
d [A]
= −kdt
[A]
[ A] d [A] t
∫[A]0 [A] = −k ∫0 dt
ln[A] − ln[A]0 = −kt
ln[A] = ln[A]0 − kt
[A] = [A]0 e − kt
(t)
The reactant concentration
decreases exponentially with time
Half-life is constant for a first-order reaction

ln[A] − ln[A]0 = −kt

[A]
ln = −kt
[A]0
1
ln = −kt 1 / 2
2
ln 2 0.693
t1 / 2 = =
k k
For the first-order reaction, half-life is independent of
the initial reactant concentration
Second-order reaction with one reactant
2A → P
d [A]
v=− = k [ A ]2
dt
d [A] 1 Slope= k
= −kdt
[A] 2 [A]
[ A] d [A] t
∫[A]0 [A]2 = − k ∫0 dt
1
1 1
= + kt [A]0
[A] [A]0
Time (t)
2 1 1
kt 1 / 2 = − =
[A]0 [A]0 [A]0
1
t1 / 2 = Half-life is dependent on the initial reactant concentration
k[A]0
Pseudo first-order reactions

v = k[A][B]
When [B] >> [A],
v = k ′[A]
where k ′ = k[B]0

e.g. B is water (55.5M): k'= 55.5k

Arrhenius equation
k = Ae-Ea/RT

= Activation energy (Ea)

 Multistep reactions have
rate-determining steps

k1 > k2

k1 < k2

k1 k2

Rate-determining step: the slow step (= the higher activation energy)

Catalysts reduce the activation energy

ΔEa
(the reduction
in Ea by the
catalyst)

Rate enhancement = kcat/kuncat = e ΔEa/RT

e.g. When ΔEa = 5.7 kJ/mol, the reaction rate increases 10 fold
When ΔEa = 34 kJ/mol, the reaction rate increases 106 fold
Michaelis-Menten equation

Vmax [S]
v0 =
K M + [S]
• v0 = the initial rate
• Vmax = the maximum rate
• KM = the Michaelis constant
• [S] = the substrate concentration
Steady state approximation
k1 k2
E+S ES P+E
k-1
Derivatization of Michaelis-Menten equation
k1 k2
E+S ES P+E
k-1
d [ P]
v= = k 2[ES]
dt
d [ES]
= k 1[E][S] − k − 1[ES] − k 2[ES] = 0 (Steady state approximation)
dt
[E]T = [E] + [ES]
k 1([E]T − [ES])[S] = (k − 1 + k 2)[ES]
(k − 1 + k 2 + k 1[S])[ES] = k 1[E]T[S]
k − 1 + k2
( + [S])[ES] = [E]T[S]
k1
[E]T[S]
[ES] =
k − 1 + k2
+ [S]
k1
[E]T[S] k − 1 + k2
[ES] = where KM =
KM + [S] k1
k 2[E]T[S]
The initial velocity v 0 = k 2[ES] =
KM + [S]
The maximum velocity (Vmax) occurs at high substrate concentration
when the enzyme is entirely in the [ES] form : Vmax = k 2[E]T
Vmax [S]
v0 =
K M + [S]
 Michaelis constant KM
KM = [S] at which v0 = Vmax/2

• If an enzyme has a small value of KM, it achieves maximal catalytic efficiency

at low substrate concentrations
• Measure of the enzyme’s binding affinity for the substrate
(The lower KM, the higher affinity)
 Lineweaver-Burke plot

Vmax [S]
v0 =
K M + [S]
1 K M + [S]
=
v 0 Vmax [S]
1 ⎛ KM ⎞ 1 1
= ⎜⎜ ⎟⎟ +
v 0 ⎝ Vmax ⎠ [S] Vmax
kcat/KM is a measure of catalytic efficiency

Catalytic constant or turnover number of an enzyme :

Vmax
kcat =
[E]T

When KM >> [S],

very little ES is formed and consequently [E] ≈ [E]T
Vmax[S] Vmax[S] kcat[E]T[S] ⎛ kcat ⎞
v0 = ≈ = ≈ ⎜ ⎟[E][S]
KM + [S] KM KM ⎝ KM ⎠
 Catalytic perfection
kcat k 2 k 1k 2
= =
KM K M k − 1 + k 2
The ratio is maximal when k 2 >> k − 1,
kcat
= k1
KM
(Diffusion-controlled limit: 108 to 109 M-1s-1)
 Inhibitors
• Substances that reduce an enzyme’s activity
– Study of enzymatic mechanism
– Therapeutic agents
• Reversible or irreversible inhibitors

O CO2-
O CO2-
O N CO2-
H NH2 N CO2-
N H
HN N N
N N
H
H2N N N CH3
H2N N N
H

Dihydrofolate Methotrexate
(Dihydrofolate reductase substrate) (Dihydrofolate reductase inhibitor,
anticancer drug )
 Modes of the reversible inhibition
• Competitive inhibitors
– Binds to the substrate
binding site
• Uncompetitive inhibitors
– Binds to enzyme-
substrate complex
• Non-competitive inhibitors
– Binds to a site different
from the substrate
binding site
• Mixed inhibitors
– Binds to the substrate-
binding site and the
enzyme-substrate
Competitive inhibition
[E][I]
KI =
[EI]
d [ES]
= k 1[E][S] − k − 1[ES] − k 2[ES] = 0 (Steady state approximation)
dt
⎛ k − 1 + k 2 ⎞ [ES] KM [ES]
[E] = ⎜ ⎟ =
⎝ k 1 ⎠ [S] [S]
[E][I] KM [ES][I]
[EI] = =
KI [S]KI
[E]T = [E] + [EI] + [ES]
KM [ES] KM [ES][I] ⎧ KM ⎛ [I] ⎞ ⎫
[E]T = + + [ES] = [ES]⎨ ⎜1 + ⎟ + 1⎬
[S] [S]KI ⎩ [S] ⎝ KI ⎠ ⎭
[E]T[S]
[ES] =
⎛ [I] ⎞
KM ⎜1 + ⎟ + [S]
⎝ KI ⎠
k 2[E]T[S]
v 0 = k 2[ES] =
⎛ [I] ⎞
KM ⎜1 + ⎟ + [S]
⎝ KI ⎠
Since Vmax = k 2[E]T,
Vmax [S] [I]
v0 = where α = 1 +
αK M + [S] KI
Competitive inhibitors affect KM
Vmax [S]
v0 =
αK M + [S]
[S] → ∞; v0 → Vmax regardless of α
 Determination of KI
of the competitive inhibitor
1 ⎛ α KM ⎞ 1 1
=⎜ ⎟ +
v0 ⎝ V max ⎠ [S] V max
Uncompetitive inhibition
[ES][I]
KI ' =
[ESI]
KM [ES]
[E] =
[S]
[ES][I]
[ESI] =
KI '
[E]T = [E] + [ES] + [ESI]
KM [ES] [ES][I] ⎛ KM [I] ⎞
[E]T = + [ES] + = [ES]⎜⎜ + 1 + ⎟⎟
[S] KI ' ⎝ [S] KI ' ⎠
[E]T[S]
[ES] =
⎛ [I] ⎞
KM + ⎜1 + ⎟[S]
⎝ KI ' ⎠
k 2[E]T[S]
v 0 = k 2[ES] =
⎛ [I] ⎞
KM + ⎜1 + ⎟[S]
⎝ KI ' ⎠
Since Vmax = k 2[E]T,
Vmax
[S]
Vmax [S] α ' [I]
v0 = = where α ' = 1 +
K M + α '[S] K M + [S] KI '
α'
 Uncompetitive inhibitors
decrease both Vmax and KM
Vmax
[S]
v0 = α '
KM
+ [S]
α'
Vmax
[S] → ∞; v0 →
α'
V (Effects of an uncompetitive inhibitor
K M >> [S]; v0 → max [S]
KM become negligible)

Vmax
no inhibitor (α'=1)
Increasing [I]
VmaxI

v0 Vmax/2
[I]
VmaxI/2 α ' = 1+
KI '

KMI KM [S]
 Determination of KI’
of the uncompetitive inhibitor
1 ⎛ KM ⎞ 1 α'
=⎜ ⎟ +
v0 ⎝ V max ⎠ [S] V max
Mixed inhibition
[E][I] [ES][I]
KI = KI ' =
[EI] [ESI]
[E][I]
[EI] =
KI
[ES][I]
[ESI] =
KI '
[E]T = [E] + [EI] + [ES] + [ESI]
⎛ [I] ⎞ ⎛ [I] ⎞
[E]T = [E]⎜1 + ⎟ + [ES]⎜1 + ⎟
⎝ KI ⎠ ⎝ KI ' ⎠
[E]T = [E]α + [ES]α '
KM [ES] ⎛ αKM ⎞
[E]T = α + [ES]α ' = [ES]⎜⎜ + α ' ⎟⎟
[S] ⎝ [S] ⎠
[E]T[S]
[ES] =
αKM + α '[S]
k 2[E]T[S]
v 0 = k 2[ES] =
αKM + α '[S]
Since Vmax = k 2[E]T,
Vmax [S] [I] [I]
v0 = where α = 1 + and α ' = 1 +
αK M + α '[S] KI KI '
Lineweaver-Burk plot of a mixed inhibition
1 ⎛ αKM ⎞ 1 α'
=⎜ ⎟ +
v0 ⎝ V max ⎠ [S] V max
Noncompetitive inhibition
A special mixed inhibition when KI = KI’

Vmax [S] [I] [I]

v0 = where α = 1 + and α ' = 1 +
αK M + α '[S] KI KI '
When KI = KI' , α = α '
Vmax
[S]
Vmax [S]
v0 = = α
α ( K M + [S]) K M + [S]
Noncompetitive inhibitors affect not KM but Vmax
Vmax
[S]
[ I]
v0 = α where α = 1 +
K M + [S] KI
Vmax
[S] → ∞; v0 →
α

Vmax

no inhibitor (α=1)
Vmax I Increasing [I]

v0 Vmax/2
VmaxI/2 [I]
α = 1+
KI

KM [S]
 Determination of KI
of the noncompetitive inhibitor
1 ⎛ α KM ⎞ 1 α
=⎜ ⎟ +
v0 ⎝ V max ⎠ [S] V max
1/v0

α=2

Increasing α= 1.5
[I]

α= 1
(no inhibitor)

Slope=αKM/Vmax
[ I]
α/Vmax α = 1+
KI
1 0 1/[S]
−
KM
Effects of inhibitors on Vmax and KM
of the Michaelis-Menten equation

app
Vmax [S]
v0 = app
K M + [S]
 Effects of pH
'
Vmax [S]
• Binding of substrate to enzyme v0 = '
K M + [S]
• Catalytic activity of enzyme '
Vmax = Vmax / f 2 and K M' = K M ( f 1 / f 2)
• Ionization of substrate where

• Variation of protein structure [H + ] KE2

f1= +1+ +
KE1 [H ]
(only at extreme pHs)
[H + ] KES2
f2= +1+ +
KES1 [H ]
E- ES-

KE2 H+ KES2 H+
k1 k2
EH + S ESH P + EH
k-1
KE1 H+ KES1 H+

EH2+ ESH2+
Approximate identity of catalytic amino acid residues

pKa ~4 → Catalytic Asp or Glu residue

pKa ~6 → Catalytic His residue
pKa ~10 → Catalytic Lys residue
Caution should be taken because pKa of amino acid residues are environmentally sensitive
 Bisubstrate reactions

• 60% of biochemical reactions involve two substrates and two products

• Transfer reactions and oxidation-reduction reactions
 Cleland’s nomenclature system
for the enzymatic reactions

• Substrates: A, B, C, D… in the order that they add to the enzyme

• Products: P, Q, R, S… in the order that they leave the enzyme
• Inhibitors: I, J, K, L…
• Stable enzyme complexes: E, F, G, H… with E being the free
enzyme
• Numbers of reactants and products: Uni (one), Bi (two), Ter (three),
and Quad (four)
e.g. Bi Bi reaction: a reaction that requires two substrates and yields
two products
 Types of Bi Bi Reactions
• Sequential reactions (single displacement reactions):
all substrates bind before chemical event

Ordered mechanism

Random mechanism

• Ping pong reactions (double displacement reactions):

chemistry occurs prior to binding of all substrates
Differentiating bisubstrate mechanisms
• Measure rates
• Change concentration of substrates and
products

• Lineweaver-Burk plot
– Intercept (1/Vmax): the velocity at saturated substrate
concentration → It changes when the substrate A
binds to a different enzyme form with the substrate B
– Slope (KM/Vmax): the rate at low substrate
concentration → It changes when both A and B
reversibly bind to an enzyme form
 Ping Pong Bi Bi Mechanism

• Intercept changes because A and B bind to the different enzyme forms E and
F, respectively
• Slope remains same because the binding of A and B is irreversible due to the
release of the product (P)
 Sequential Bi Bi Mechanism

or

Ordered
Random
• Intercept changes because A and B binds to the different enzyme forms
(E or EB) and (EA or E), respectively
• Slope changes because the binding of A and B is reversible
 Differentiating Bi Bi mechanisms
by product inhibition
Competitive inhibition → Substrate and inhibitor competitively bind to the same
site of the enzyme

A vs Q: Competitive
B vs P: Competitive
Ping Pong A vs P: Noncompetitive
B vs Q: Noncompetitive

A vs Q: Competitive
B vs P: Noncompetitive
Ordered A vs P: Noncompetitive
sequential
B vs Q: Noncompetitive

Under assumption of dead-end complex formation

(A is similar with Q and B is similar with P)
Random A vs Q: Competitive
sequential B vs P: Competitive
A vs P: Noncompetitive
B vs Q: Noncompetitive
 Dead-end complexes
A B

Q P

Q B Dead-end complex (no chemistry)

A P no chemistry

competitive

ATP + Creatine ADP + Creatine-phosphate

competitive
Differentiating Bi Bi mechanisms
by isotope exchange

Ping Pong Mechanism

A*→ P isotope exchange is possible without B
B* → Q isotope exchange is possible without A

or

Sequential Mechanism
Two substrates are required for the isotope exchange
 Isotope exchanges in a ping pong mechanism

Sucrose phosphorylase
(Sucrose) E
Glucose-fructose + phosphate Glucose-1-phosphate + fructose

Isotope exchange experiments

E
Glucose-fructose + fructose* Glucose-fructose* + fructose
E
Glucose-1-phosphate + phosphate* Glucose-1-phosphate* + phosphate

E
Glucose-fructose + E Fructose + Glucose-E
E
Glucose-E + phosphate Glucose-1-phosphate + E

Ping Pong Mechanism (Double displacement)

 Isotope exchanges in a sequential mechanism

Maltose phosphorylase
(Maltose)
E
Glucose-glucose + phosphate Glucose-1-phosphate + glucose

Isotope exchange experiments

E
Glucose-glucose + glucose* No isotope exchange
E
Glucose-1-phosphate + phosphate* No isotope exchange
E
Glucose-glucose + phosphate* Glucose-1-phosphate* + glucose
E
Glucose-1-phosphate + glucose* Glucose-glucose + phosphate

Sequential Mechanism (Single displacement)