Kinetics of enzyme catalyzed

reactions

1
 Enzyme catalysis
• Compounds within the cell can be manufactured at specified
reaction rates
• A cell can select which reactants will be combined and which
can be decomposed.
• How does this occur?
Catalysis by enzymes or globular proteins

Isolated enzymes enjoy a wide spectrum of applications

2
Enzyme Commission classification system for
enzymes
1. Oxidoreductases (Oxidation-reduction reactions)
2. Transferases (transfer of functional groups)
3. Hydrolases (hydrolysis reactions)
4. Lyases (addition to double bonds)
5. Isomerases (isomerization reactions)
6. Ligases (formation of bonds with ATP cleavage)

3
Microbial Enzymes in Food Applications

4
Microbial Enzymes in Food Applications

5
 Activation energy for reaction
A catalyst influences the rate of reaction with affecting the
equilibrium

Uncatalyzed reaction Catalyzed reaction 6

 Characteristic Features
• Specific
• Requires cofactors
• Higher turnover number (net number of substrate molecules
reacted per catalyst site per unit time) at ambient temperature

Enzyme activity: Moles of substrate converted per unit time ( 𝑟𝑎𝑡𝑒 ×

𝑣𝑜𝑙𝑢𝑚𝑒)
Enzyme Unit: The amount of the enzyme that catalyzes the conversion of
one μmol of substrate per minute under the standard conditions
7
 Cofactor
Non protein compound which combines with inactive protein
(apoenzyme) to give a catalytically active complex (holoenzyme
or enzyme)
• Metal ions
• Complex organic molecules like NAD, FAD, coenzyme A may
be a cofactor
Enzymes with the same name but obtained from different
organisms: have different amino acid sequences, thus having
different properties and catalytic activities
8
 Enzyme catalysis
• Enzyme E and substrate S combine to from a complex ES
which then dissociates into product P and free enzyme E
𝑘1
𝑆 + 𝐸 ՞ ES
𝑘−1
𝑘2
𝐸𝑆 ՜ P + E
Lock and key model

• Substrate binds to the active site of the Enzyme

• Active site includes Asp, Cys, Glu, His, Lys, Met, Ser, Thr and the end
amino and carboxyl groups 9
 Michaelis-Menton Kinetics
𝑘1
Henery (1902) and Michaelis and Menton (1913) 𝑆 + 𝐸 ՞ ES
proposed 𝑘−1
𝑣𝑚𝑎𝑥 𝑠
𝑣= 𝑘2
𝐾𝑚 + 𝑠 𝐸𝑆 ՜ P + E

• The rate of reaction is first order in concentration of substrate at

relatively low concentrations
• As the substrate concentration is continually increased, reaction
order in substrate reduces continuously from one to zero
• Rate of reaction is proportional to amount of enzyme present
10
 𝑘1
𝐸 + 𝑆 ՞ 𝐸𝑆 ՜ 𝑃 + 𝐸
𝑘2 Contd…
𝑘−1 𝐸 [𝑆]
𝐸𝑞𝑢𝑖𝑙𝑖𝑏𝑟𝑖𝑢𝑚 𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡, 𝐾𝑚 = =
𝑘1 [𝐸𝑆]
𝑑𝑃
𝑣= = 𝑘2 [𝐸𝑆]
𝑑𝑡

𝑑[𝐸𝑆]
= 𝑘1 𝐸 𝑆 − 𝑘−1 𝐸𝑆 − 𝑘2 [𝐸𝑆]
𝑑𝑡
𝐸0 = 𝐸 + 𝐸𝑆

( 𝐸0 − 𝐸𝑆 )[𝑆] 𝐸0 [𝑆]
𝐾𝑚 = ֜ [𝐸𝑆] =
[𝐸𝑆] 𝐾𝑚 + [𝑆]

𝑑𝑃 𝐸0 [𝑆] 𝑣𝑚𝑎𝑥 [𝑆]

𝑣= = 𝑘2 𝐸𝑆 = 𝑘2 = [𝑣𝑚𝑎𝑥 = 𝑘2 [𝐸0 ]]
𝑑𝑡 𝐾𝑚 +[𝑆] 𝐾𝑚 +[𝑆]

11
 Contd…
• Product-releasing step is
much slower than the
reversible reaction
• The slower step determines
𝑣𝑚𝑎𝑥 𝑠 the rate, while the other is at
𝑣= equilibrium
𝐾𝑚 + 𝑠

𝑆𝐸 𝑘−1
𝐾𝑚 = =
(𝐸𝑆) 𝑘1
(𝐷𝑖𝑠𝑠𝑜𝑐𝑖𝑎𝑡𝑖𝑜𝑛 𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡)
12
 Briggs and Haldane Approach
The change of the intermediate concentration with respect to time is assumed to
be negligible i.e. the pseudo-steady-state (or quasi-steady-state)
𝑘1 𝑘2 𝑘−1 + 𝑘2
𝑆 + 𝐸 ՞ ES 𝐸𝑆 ՜ P + E 𝐾𝑚 =
𝑘 𝑘1
−1

13
 Evaluation of rate parameters
𝒗𝒎𝒂𝒙 𝒔
𝒗=
𝑲𝒎 + 𝒔
Lineweaver-Bulk plot
1 1 𝐾𝑚 1
= +
𝑣 𝑣𝑚𝑎𝑥 𝑣𝑚𝑎𝑥 𝑠
Hanes-Woolf plot
𝑠 𝑠 𝐾𝑚
= +
𝑣 𝑣𝑚𝑎𝑥 𝑣𝑚𝑎𝑥
Eadie-Hofstee plot
𝑣
𝑣 = 𝑣𝑚𝑎𝑥 − 𝐾𝑚
𝑆
14
 Contd…
Lineweaver-Bulk plot

1 1 𝐾𝑚 1
= +
𝑣 𝑣𝑚𝑎𝑥 𝑣𝑚𝑎𝑥 𝑠

Data points at low substrate

concentrations influence the
slope and intercept

15
Eadie-Hofstee plot Contd…
𝑣
𝑣 = 𝑣𝑚𝑎𝑥 − 𝐾𝑚
𝑆

Subject to large errors since both axes

contain measured variable 𝑣

16
 Contd…
Hanes-Woolf plot

𝑠 𝑠 𝐾𝑚
= +
𝑣 𝑣𝑚𝑎𝑥 𝑣𝑚𝑎𝑥

This plot is used to determine

𝑣𝑚𝑎𝑥 more accurately

17
 Evaluation of rate parameters
𝒗𝒎𝒂𝒙 𝒔
𝒗=
𝑲𝒎 + 𝒔
Analytically,
𝒅𝒔 𝒗𝒎𝒂𝒙 𝒔
=
𝒅𝒕 𝑲𝒎 + 𝒔
𝒅𝒔 𝑲𝒎
𝟏+ = 𝒗𝒎𝒂𝒙
𝒅𝒕 𝒔
After Integration
𝒔𝟎
𝑲𝒎 𝒍𝒏 + (𝒔𝟎 −𝒔) = 𝒗𝒎𝒂𝒙 × 𝒕
𝒔

18
 Enzyme Inhibition
• Competitive Inhibition 𝑘1 𝑘2
𝑆 + 𝐸 ՞ ES ՜ 𝑃 + 𝐸
𝑘−1
• Noncompetitive Inhibition

• Uncompetitive Inhibition

19
 Competitive Inhibition
Inhibitor binds only to the free enzyme, not to the ES complex

E+S ES E+P
+
I

EI

20
 Contd…
𝑲𝒔 𝒗𝒎𝒂𝒙 𝒔
𝒌 𝒗=
E+S ES E+P [𝑰]
𝑲𝒔 𝟏+ +𝒔
+ 𝑲𝒊
I 𝒗𝒎𝒂𝒙 𝒔
𝒗 = 𝒂𝒑𝒑
𝑲𝑰 𝑲 𝒎+𝒔
EI [𝑰]
𝑲𝒂𝒑𝒑 𝒎 = 𝑲𝒔 𝟏+
𝑲𝒊

21
 Noncompetitive Inhibition
Inhibitor binds enzyme regardless of whether the substrate is bound
𝒌
E+S ES E+P
+ 𝑲𝒔 + 𝒗𝒂𝒑𝒑𝒎𝒂𝒙 𝒔
𝒗=
𝑲𝒔 + 𝒔
I I
𝑲𝑰
𝒌𝑬𝟎
EI + S ESI 𝒗𝒂𝒑𝒑 𝒎𝒂𝒙 =
[𝑰]
𝟏+
𝑲𝒊
22
 Uncompetitive Inhibition
Inhibitor binds only to the ES complex and not to the free enzyme
𝑲𝒔 𝒌 𝒗𝒂𝒑𝒑 𝒔𝒎𝒂𝒙
E+S ES E+P 𝒗=
𝑲𝒂𝒑𝒑𝒎 + 𝒔
+
I 𝑲𝒂𝒑𝒑 =
𝑲𝒔
𝒎
[𝑰]
𝑲𝑰 𝟏+
𝑲𝒊 𝒔
𝒌𝑬𝟎
ESI 𝒗𝒂𝒑𝒑 𝒎𝒂𝒙 =
[𝑰]
𝟏+
𝑲𝒊
23
Lineweaver-Bulk plot for inhibition

24
 Reversible reactions
𝑘1 𝑘2
𝑆 + 𝐸 ՞ ES 𝐸𝑆 ՞ P + E
𝑘−1 𝑘−2

25
 Two-substrate reactions
Dissociation Equ. Const.
𝐸 + 𝑆1 ՞ 𝐸𝑆1 𝐾1
𝑘𝐸0
𝐸 + 𝑆2 ՞ 𝐸𝑆2 𝐾2 𝑣=
𝐾21 𝐾12 1/2(𝐾2 𝐾21 + 𝐾1 𝐾12 )
𝐸𝑆1 + 𝑆2 ՞ 𝐸𝑆1 𝑆2 𝐾12 1+
𝑆1
+
𝑆2
+
𝑆1 𝑆2
𝐸𝑆2 + 𝑆1 ՞ 𝐸𝑆1 𝑆2 𝐾21
𝑇ℎ𝑒 𝑒𝑞𝑢𝑖𝑙𝑖𝑏𝑟𝑖𝑢𝑚 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑠 𝐾2 𝐾21 = 𝐾1 𝐾12
𝑘
𝐸𝑆1 𝑆2 ՜ 𝑃 + 𝐸 ∗
𝑘𝐸0 𝑆2
∗
𝑣𝑚𝑎𝑥 𝑆1 𝑣𝑚𝑎𝑥 =
𝑣 = 𝑘[𝐸𝑆1 𝑆2 ] 𝑣= ∗ 𝑆2 + 𝐾12
𝐾1 + 𝑆1
∗ 𝐾21 𝑆2 + 𝐾1 𝐾12
𝐾1 =
𝑆2 + 𝐾1226
 Contd…

𝑣𝑚𝑎𝑥 ∗ 𝑆1 ∗
𝑘𝐸0 𝑆2
𝑣= ∗ 𝑣𝑚𝑎𝑥 =
𝐾1 + 𝑆1 𝑆2 + 𝐾12

∗ 𝐾21 𝑆2 + 𝐾1 𝐾12
𝐾1 =
𝑆2 + 𝐾12
𝐼𝑓 𝑆2 ≫ 𝐾12 𝑘𝐸0 𝑆1
𝑣𝑚𝑎𝑥 ∗ = 𝑘𝐸0 𝑣=
𝐾21 + 𝑆1
𝐾1 ∗ ≈ 𝐾21

27
Mechanism with a cofactor

• Resembles closely the two-

substrate mechanism (𝑆2 ≫ 𝐾12 )

𝑘𝐸0 𝑐𝑆
𝑣=
𝐾𝑠 (𝑐 + 𝐾𝑐 ) + 𝑐𝑆

Where c is the cofactor concentration

28
 Substrate Inhibition
𝐸 + 𝑆 ՞ 𝐸𝑆 𝐾1 Dissociation 𝑘𝐸0
𝐸𝑆 + 𝑆 ՞ 𝐸𝑆2 𝐾2 𝑣=
𝑘
Constant 1 + 𝐾1 /𝑆 + 𝑆/𝐾2
𝐸𝑆 ՜ 𝑃 + 𝐸
𝑆𝑚𝑎𝑥 = 𝐾1 𝐾2 (𝑑𝑣/𝑑𝑠 = 0)

Inhibition
Activation

29
 Other factors
• pH
• Temperature
• Fluid forces
• Chemical agents
• Irradiation

30
 Strategies for Enzyme Stabilization
• Adding stabilizing compounds
• Chemical modification
• Immobilization

31
Tutorial

32
 Problem-1
• The hydrolysis of urea by urease is an only partially understood
reaction and shows inhibition.
Substrate concentrations

a. Determine the Michaelis–Menten

constant (Km) for this reaction.
b. What type of inhibition reaction is
this?
c. Based on the answer to part b, what is
the value of KI?

where v = moles/l-min and I is inhibitor molar concentration. 33

34
 Problem-2
Given Michaelis-Menten kinetics for a concentration of enzyme
[E], assume the Km value of 6.2 mmol ml-1. The initial and final
substrate concentrations were 14 and 0.2 mmol ml-1, respectively.
Also given is the maximum rate for the enzyme Vmax 5.6 µmol
(ml min)-1.

Determine batch reaction time for the given information about the
enzyme kinetic activities.
𝒔𝟎
𝑲𝒎 𝒍𝒏 + (𝒔𝟎 −𝒔) = 𝒗𝒎𝒂𝒙 × 𝒕
𝒔
35
 Problem-3
Lipase is being investigated as an additive to laundry detergent for
removal of stains from fabric. The general reaction is:
fats՜fatty acids + glycerol
The Michaelis constant for pancreatic lipase is 5 mM. At 60°C, lipase
is subject to deactivation with a half-life of 8 min. Fat hydrolysis is
carried out in a well-mixed batch reactor that simulates a top-loading
washing machine. The initial fat concentration is 45 gmol m-3. At the
beginning of the reaction, the rate of hydrolysis is 0.07 mmol l-1 s-1.

How long does it take for the enzyme to hydrolyse 80% of the fat
present?
𝟏 𝑲𝒎 𝒔𝟎
𝒕 = − 𝒍𝒏 𝟏 − 𝒍𝒏 + (𝒔𝟎 −𝒔)
𝒌𝒅 𝒗𝒎𝒂𝒙 𝒔 36