Enzyme kinetics

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 Example 1
Statins  competitive inhibitors of HMG-CoA reductase

(3-hydroxy-3-methylglutaryl-coenzyme A)

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Statins  competitive inhibitors of HMG-CoA reductase
HMG-CoA
Statins
HMG-CoA
reductase
Statins

HMG-CoA
reductase

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Example 2

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 Enzymes

 Enzymes are biocatalysts

 Enzyme are usually proteins of high molecular weight (15000 <MW < several million daltons)

 Some enzymes require a non-protein groups for their activity, a co-actor (Mg, Zn, Mn etc.,)
or a co-enzyme (NAD, FAD or some vitamins)

 Isozymes: Enzymes that occur in several different molecular forms, but catalyze the same reaction

 Enzymes are substrate specific and are classified according to the reactions they catalyze

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 Enzymes lower the activation energy of the
reaction catalyzed by binding the substrate and
forming an enzyme substrate complex

 Enzymes do not affect the free energy change

or the equilibrium constant

 Weak interactions between enzyme and substrate

(van der walls forces or hydrogen bonding)

 Substrate binds to specific region of the enzyme called active site (Lock and key model)

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Enzyme kinetics for single-substrate-enzyme-catalyzed reactions

 Often referred to as Michaelis-Menton kinetics or saturation kinetics

 Qualitative features or much similar to Langmuir-Hinshelwood kinetics

 Models are based on the data from batch reactors with constant liquid volume, with
known initial substrate and enzyme concentrations

Reaction scheme for saturation kinetics

k1 k2
E+S ES E+P
k-1

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Mechanistic models for simple enzyme kinetics
Rate of product formation is

𝑑[𝑃]
𝑣= = 𝑘2 [𝐸𝑆] (1)
𝑑𝑡
The rate of change of ES is given by

𝑑[𝐸𝑆]
= 𝑘1 𝐸 𝑆 − 𝑘−1 𝐸𝑆 − 𝑘2 [𝐸𝑆] (2)
𝑑𝑡
Total enzyme at any time is given by

𝐸0 = 𝐸 + [𝐸𝑆] (3)

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The rapid equilibrium assumption – Michaelis-Menton approach

Assuming a rapid equilibrium is established between enzyme and substrate to form

an ES complex

The equilibrium constant is

′
𝑘−1 𝐸 [𝑆]
𝐾𝑚 = = (4)
𝑘1 [𝐸𝑆]

Substituting 𝐸 = 𝐸0 − [𝐸𝑆]

𝐸0 [𝑆]
𝐸𝑆 = (5)
𝑘−1 /𝑘1 + [𝑆]

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 𝐸0 [𝑆]
𝐸𝑆 = ′ (6)
𝐾𝑚 + [𝑆]

𝑑[𝑃] 𝐸0 [𝑆] 𝑉𝑚 [𝑆]

𝑣= = 𝑘2 ′ = ′ (7)
𝑑𝑡 𝐾𝑚 + [𝑆] 𝐾𝑚 + [𝑆]

where vm= k2 E0
Vm maximum forward velocity of the reaction
Prime in K’m denotes rapid equilibrium approach

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The quasi-steady state assumption – Briggs-Haldane approach

 In many cases rapid equilibrium, assumption is not valid

 When the rate of product formation (k2) from the ES complex is comparable
to the rate of breakdown (k-1) of ES complex to enzyme and substrate

 Quasi steady-state assumption: [S0]>>[E0], the intermediate complex ES is at steady state

𝑑[𝐸𝑆]
≈0 (8)
𝑑𝑡

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Flaw in quasi-steady state assumption

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 𝑑[𝐸𝑆]
Apply QSS in Eq (2) = 𝑘1 𝐸 𝑆 − 𝑘−1 𝐸𝑆 − 𝑘2 𝐸𝑆 = 0 (9)
𝑑𝑡

𝑘1 [𝐸] [𝑆]
𝐸𝑆 = (10)
𝑘−1 + 𝑘2

𝑘1 𝐸0 − 𝐸𝑆 [𝑆]
𝐸𝑆 = (11)
𝑘−1 + 𝑘2

Solving the above equation for [ES]

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 𝐸0 [𝑆]
𝐸𝑆 = (12)
𝑘−1 + 𝑘2
+ [𝑆]
𝑘1

𝑘2 𝐸0 [𝑆]
𝑣 = 𝑘2 𝐸𝑆 = (13)
𝑘−1 + 𝑘2
+ [𝑆]
𝑘1

𝑉𝑚 [𝑆]
𝑣= (14)
𝐾𝑚 + [𝑆]

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Experimentally determining rate parameters for Michaelis–Menten type kinetics

 Km and Vm determination with high precision difficult

 Experimental data obtained from initial-rate experiments

 Plot of substrate concentration/product concentration versus

time give v (repeat for different S)

 Accurate determination of Km from this plot difficult

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Double-reciprocal plot (Lineweaver–Burk plot).

𝑉𝑚 [𝑆]
𝑣= (14)
𝐾𝑚 + [𝑆]

Inverse of above equation

1 1 𝐾𝑚 1
= + (15)
𝑣 𝑉𝑚 𝑉𝑚 [𝑆]

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Eadie–Hofstee plot

Rearranging eq (14)

𝑣
𝑣 = 𝑉𝑚 − 𝐾𝑚 (16)
[𝑆]

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Hanes–Woolf plot

Rearranging eq (14)

[𝑆] 𝐾𝑚 1
= + [𝑆] (17)
𝑣 𝑉𝑚 𝑉𝑚

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Batch kinetics

The time course of variation of [S] in a batch enzymatic reaction can be determined from

𝑑𝑆 𝑉𝑚 [𝑆]
𝑣=− = (18)
𝑑𝑡 𝐾𝑚 + [𝑆]

integrating

1 𝑆0
𝑡𝑏 = ( 𝑆0 − 𝑆 + 𝐾𝑚 𝑙𝑛 ) (19)
𝑉𝑚 𝑆

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With deactivation of enzymes

𝑑𝑆 𝑉𝑚 𝑒 −𝑘𝑑 𝑡 [𝑆]
𝑣=− = (20)
𝑑𝑡 𝐾𝑚 + [𝑆]

integrating

1 𝐾𝑚 𝑆0 𝑆0 − 𝑆
𝑡𝑏 = − ln[1 − 𝑘𝑑 𝑙𝑛 + ] (21)
𝑘𝑑 𝑉𝑚 𝑆 𝑉𝑚

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Inhibited enzyme kinetics

 Certain compounds many bind to enzyme and reduce the activity- enzyme inhibitors

 Reversible inhibitors: dissociate more easily from enzymes after binding

 Irreversible inhibitors: form stable complex with enzyme (heavy metals like Pb, Cd, Hg)
can be reversed using chelating agents

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Types of reversible enzyme inhibitions

a) competitive

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b) Non-competitive

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c) Uncompetitive

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d) Substrate

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a) competitive

 Competitive inhibitors are usually substrate analogs

 Compete with the enzyme for active site

Assuming rapid equilibrium

′
𝐸 [𝑆] 𝐸 [𝐼]
𝐾𝑚 = (1) 𝐾𝐼 = (2)
[𝐸𝑆] [𝐸𝐼]

𝐸0 = 𝐸 + 𝐸𝑆 + [𝐸𝐼] (3)

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 𝐸0 = 𝐸 + 𝐸𝑆 + [𝐸𝐼] (3)

′
𝐾𝑚 [𝐸𝑆] 𝐸 [𝐼]
𝐸0 = + 𝐸𝑆 + (4)
[𝑆] 𝐾𝐼

′ ′
𝐾𝑚 [𝐸𝑆] 𝐾𝑚 𝐸𝑆 [𝐼]
𝐸0 = + 𝐸𝑆 + (5)
[𝑆] 𝑆 𝐾𝐼

𝐸0 [𝑆]
𝐸𝑆 = (6)
Rearranging for [ES] ′ 𝐼
𝐾𝑚 1+ + [𝑆]
𝐾𝐼

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 𝑘2 [𝐸0 ][𝑆]
𝑣 = 𝑘2 𝐸𝑆 = (7)
′ 𝐼
𝐾𝑚 1+ + [𝑆]
𝐾𝐼

𝑉𝑚 [𝑆]
𝑣= ′ (8)
𝐾𝑚,𝑎𝑝𝑝 + [𝑆]

′ ′ 𝐼
;where 𝐾𝑚,𝑎𝑝𝑝 = 𝐾𝑚 1 +
𝐾𝐼

 Increase in Km decreases the reactions rate

 Competitive inhibition can be overcome by high concentrations of substrate

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b) Non-competitive

 Noncompetitive inhibitors are not substrate analogs

 bind on sites other than the active site and reduce
enzyme affinity to the substrate

Assuming rapid equilibrium

′
𝐸 [𝑆] 𝐸𝐼 [𝑆] 𝐸 [𝐼] 𝐸𝑆 [𝐼]
𝐾𝑚 = = (1) 𝐾𝐼 = = (2)
[𝐸𝑆] [𝐸𝑆𝐼] [𝐸𝐼] [𝐸𝑆𝐼]

𝐸0 = 𝐸 + 𝐸𝑆 + 𝐸𝐼 + [𝐸𝑆𝐼] (3) 𝑣 = 𝑘2 [𝐸𝑆] (4)

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 From (1), (2), (3) and (4) we get

𝑉𝑚
𝑣= ′ (5)
[𝐼] 𝐾𝑚
1+ 1+
𝐾𝐼 [𝑆]

𝑉𝑚,𝑎𝑝𝑝 𝑉𝑚,𝑎𝑝𝑝 [𝑆]

𝑣= ′ = ′ (6)
𝐾𝑚 𝐾𝑚 + [𝑆]
1+
[𝑆] where 𝑉𝑚,𝑎𝑝𝑝 =
𝑉𝑚
𝐼
1+𝐾
𝐼

 Reduction in Vm

 Higher substrate concentrations cannot overcome non competitive inhibition

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c) Uncompetitive

 Uncompetitive inhibitors bind to the ES complex only

 No affinity for the enzyme itself

′
𝐸 [𝑆] 𝐸𝑆 [𝐼]
𝐾𝑚 = (1) 𝐾𝐼 = (2)
[𝐸𝑆] [𝐸𝑆𝐼]

𝐸0 = 𝐸 + 𝐸𝑆 + [𝐸𝑆𝐼] (1) 𝑣 = 𝑘2 [𝐸𝑆] (4)

Solving (1), (2), (3) for [ES] and substituting in (4) we get

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 𝑉𝑚 [𝑆]
[𝐼[
1+
𝐾𝐼
𝑣= ′ (5)
𝐾𝑚
+ 𝑆
𝐼
1+
𝐾𝐼

𝑉𝑚,𝑎𝑝𝑝 [𝑆]
𝑣= (6)
𝐾𝑚,𝑎𝑝𝑝 + [𝑆]

 reduction in both V’m and K’m values.

 reduction in V’m has a more pronounced effect than the reduction in K’m , and the net
result is a reduction in reaction rate.
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Problem # 34:
The enzyme, fumarase, has the following kinetic constants:
k1 k2
E+S ES E+P
k-1

where
k1 = 109 M-1 s-1
k-1 = 4.4 x 104 s-1
k2 = 103 s-1
a. What is the value of the Michaelis constant for this enzyme?
b. At an enzyme concentration of 10-6 M, what will be the initial rate of product formation at a
substrate concentration of 10-3 M?

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In an enzyme-catalyzed reaction, the following data were recorded

a. Determine the Michaelis–Menten

constant for the reaction with no
inhibitor present at 30°C and at 49.6°C.
b. Determine the maximum velocity of
the uninhibited reaction at 30°C and
an enzyme concentration of 1.6 g/l.
c. Determine the KI for the inhibitor at
30°C and decide what type of inhibitor
is being used.

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Problem # 35

An inhibitor (I) is added to the enzymatic reaction at a level of 1.0 g/l. The following data
were obtained for Km = 9.2 g S/l.

a. Is the inhibitor competitive or noncompetitive?

b. Find KI.

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