Chapter 9.

Enzymatic Reaction Fundamentals

Dr. Endah Retno Dyartanti
Chemical Engineering Departement
Universitas Sebelas Maret
 Enzymes
Michaelis-Menten Kinetics
Enzymes are protein-like substances with catalytic properties.

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 Enzymes
Enzymes provide a pathway for the substrate to
proceed at a faster rate. The substrate, S, reacts
to form a product P.

S Slow P

E•S
Fast

A given enzyme can only catalyze only one reaction.

Example, Urea is decomposed by the enzyme urease.
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 Enzymes - Urease
A given enzyme can only catalyze only one reaction. Urea is
decomposed by the enzyme urease, as shown below.

NH2CONH2 + UREASE H→


2O
2NH3 + CO2 + UREASE
S + E →
 P+E
H 2O

The corresponding mechanism is:

E + S k1
→ E •S
E • S  → E + S
k2

E • S + W  → P + E
k3

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 Enzymes - Michaelis-
Michaelis-Menten Kinetics
rP = k3 (E • S )(W )
rE •S = 0 = k1 (E )(S ) − k2 (E • S ) − k3W (E • S )

k1 (E )(S )
(E • S ) =
k2 + k3W

Et = (E ) + (E • S )
Et
(E ) =
 k1S 
1 +  
6  k2 + k3W 
 Enzymes - Michaelis-
Michaelis-Menten Kinetics
kcat Vmax
} 678
k3W Et S kcat Et S
rP = k3 (E • S )(W ) = =
k2 + k3W K + S
+S M
k1
1424 3
KM

Vmax S
rP = k3 (E • S )(W ) =
Km + S
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 Enzymes - Michaelis-
Michaelis-Menten Kinetics
Vmax=kcatEt

Turnover Number: kcat

Number of substrate molecules (moles) converted to
product in a given time (s) on a single enzyme molecule
(molecules/molecule/time)
kcat
For the reaction: H2O2 + E →H2O + O + E

40,000,000 molecules of H2O2 converted to product per

second on a single enzyme molecule.

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 Enzymes - Michaelis-
Michaelis-Menten Kinetics
Michaelis-Menten Equation
VmaxS
rP = −rS =
KM + S
(Michaelis-Menten plot)
Vmax VmaxS1/ 2
Vmax Solving: =
2 K M + S1/ 2
-rs KM=S1/2
therefore KM is the
concentration at which the rate
9 S1/2 CS is half the maximum rate.
 Enzymes - Michaelis-
Michaelis-Menten Kinetics
1 1 KM  1 
Inverting yields: = +  
− rS Vmax Vmax  S 
Lineweaver-Burk Plot

1/-rS slope = KM/Vmax

1/Vmax

10 1/S
 Types of Enzyme Inhibition
Competitive
E + I ⇔ I • E (inactive)

Uncompetitive
E • S + I ⇔ I • E • S (inactive)

Non-competitive
E • S + I ⇔ I • E • S (inactive)
I • E + S ⇔ I • E • S (inactive)
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 Competitive Inhibition

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 Competitive Inhibition
E + S←
→
k1
 E • S →
k3
E + P
k2

E + I←
→
k4
 E • I (inactive)
k5

1) Mechanisms:
E + S → E ⋅S E ⋅S → E + S
E ⋅S → P + E E + I → E⋅I
E⋅I → E + I
rP = k 3C E⋅S
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 Competitive Inhibition
2) Rate Laws:
rE⋅S = 0 = k1CSC E − k 2 C E⋅S − k 3C E⋅S

k1CSC E CSC E
C E⋅S = =
k2 + k3 Km
k 3CSC E
rP =
Km
rI⋅E = 0 = k 4 C I C E − k 5C I⋅E
CICE k5
C I⋅ E = KI =
14 KI k4
 Competitive Inhibition
C Etot
C Etot = C E + C E⋅S + C I⋅E CE =
CS C I
1+ +
k 3C Etot CS Km KI
rP =
CIK m
K m + CS +
KI

Vmax CS
− rS =
 CI 
CS + K m 1 + 
 KI 

1 1 km  CI  1
= + 1 + 
15 − rS Vmax Vmax  K I  CS
 Competitive Inhibition
From before (no competition): 1 = 1 + K M 1
Increasing C
− rS Vmax Vmax C S
I

Competitive

1 No Inhibition Competitive
rS KM
slope = 1 1 K M  CI  1
Vmax = + 1 + 
− rS Vmax Vmax  K I  CS
1
Intercept = 1
Vmax
CS

Intercept does not change, slope increases as

16 inhibitor concentration increases
 Uncompetitive Inhibition

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 Uncompetitive Inhibition
Inhibition only has affinity for enzyme-substrate complex
→
k1

E +S E • S →
k3
P
←
k2

→
k4

I + E •S I • E • S (inactive)
←
k5

Developing the rate law:

rP = − rS = k cat (E • S )

rE • S = 0 = k1 (E )(S ) − k 2 (E • S ) − k cat (E • S ) − k 4 (I )(E • S ) + k 5 (I • E • S ) (1)

rI•E•S = 0 = k 4 (I )(E • S ) − k5 (I • E • S ) (2)
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 Uncompetitive Inhibition
Adding (1) and (2)
k1 (E )(S ) − k 2 (E • S ) − kcat (E • S ) = 0
k1 (E )(S ) (E )(S )
(E • S ) = =
k 2 + k cat KM
From (2)
k4
(I • E • S ) = (I )(E • S ) = ( I )(E • S ) (I )(E )(S )
=
k5 KI KI KM
k5
KI =
k4
kcat (E )(S )
rp = kcat (E • S ) =
19 KM
 Uncompetitive Inhibition
Total enzyme
Et = (E ) + (E • S ) + (I • E • S )

= (E )1 +
(S ) (I )(S ) 
+ 
 KM KI KM 
kcat Et (S )
rp =

K M 1 +
( S ) (I )(S ) 
+ 
 KM KI KM 
Vmax (S )
− rS = rP =
 (I ) 
K M + (S )1 + 
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 KI 
 Uncompetitive Inhibition
1 1   (I )  
= K + (S )1 +  
− rS Vmax (S )  
M
 KI 
1 KM  1  1  (I ) 
=   + 1 + 
− rS Vmax  (S )  Vmax  KI 

Slope remains the

same but intercept
changes as inhibitor
concentration is
increased

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Lineweaver-Burk Plot for uncompetitive inhibition
 Non-competitive Inhibition

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 Non-competitive Inhibition
E+S E—S P+E

+I -I -I +I
(inactive)I.E + S I.E.S (inactive)
Increasing I

1
−
rS Vmax CS
No Inhibition − rS =
 CI 
(k M + CS )1 + 
Both slope and intercept  kI 
changes
1 1  CI  kM  1  C I 
= 1 +  +  1 + 
1 − rS Vmax  kI  Vmax  CS  k I 
CS
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 Summary: Types of Enzyme Inhibition

Lineweaver–Burk plots for three types of enzyme inhibition.

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 End of Lecture

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