BBCCT-107

ENZYMES
Indira Gandhi National
Open University
School of Sciences

Block

2
ENZYME KINETICS

UNIT 4
Enzyme Kinetics 45

UNIT 5
Bisubstrate Reactions 57

UNIT 6
Enzyme Inhibition 69
 Block 2 : Enzyme Knetics
How enzyme catalytic activity is controlled? Enzyme kinetics deals with the
measurement of rate of reaction. Our understanding about the kinetics of
an enzyme will help to understand the functional information about its
catalytic mechanism, role of enzyme in metabolism and the factors that
impact its activity as well as the mechanisms of inhibition.

This block on enzyme kinetics studies—among many things will help the
learner to determine how the rate of enzyme-catalyzed reactions depends
on the concentration of the compounds directly interacting with the enzyme
and what is the highest rate achievable by the enzyme? Further questions
of interest include the catalytic mechanism in presence of two or more
substrates. How the rate of the catalyzed reaction is affected in presence of
different types of inhibitor? All related studies provide pieces of information
that serve as input data to establish a mechanistic model of the enzymatic
reaction.

Unit 4 of the block discuss the basis of the derivation of Michelis-Menten

equation for mono substrate enzyme catalyzed reaction. Since most of the
biochemical reactions involve two or more substrates, Unit 5 dwells on
bisubstrate enzyme catalyzed reactions. Enzyme activity needs to be firmly
regulated to guarantee that levels of the product do not increase to
undesired levels. This is accomplished by enzyme inhibition. Unit 6 of the
block discusses different types of enzyme inhibition-reversible and
irreversible.

Objectives:

After studying this block, you should be able to:

 determine the rate of enzyme catalyzed reaction,

 illustrate bisubstrate enzyme catalyzed reactions,

 explain the mechanism of enzyme catalysis,

 describe different types of inhibitors regulating enzyme activity.

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Unit 4 Enzyme Kinetics
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UNIT

ENZYME KINETICS

Structure
4.1 Introduction 4.3 Summary

Objectives 4.4 Terminal Questions

4.2 Enzyme Kinetics, 4.5 Answers

Michaelis-Menten Equation 4.6 Suggested Readings
Lineweaver-Burk Plot
Significance of Km and Vmax
kcat and Turnover number

4.1 INTRODUCTION
The oldest and the important approach to understand enzyme mechanisms is
the discipline known as enzyme kinetics. Kinetics is the study of reaction
rates, their quantitative measurement and a systematic study of the factors
influencing the activity of enzymes. In this unit, you will gain an insight of the
enzymatic mechanisms as well as role played by enzyme activity in regulating
metabolic pathways. Enzymes convert substrates to products through a
series of steps known as enzymatic mechanism. Therefore the effect of
substrate concentration on enzyme activity is one of key concepts in enzyme
kinetics. Several models have been proposed to explain the kinetics of enzyme
catalyzed reactions. Classical experimental work for single enzyme catalysed
reactions is Henri-Michaelis-Menten plot, Briggs Haldane equation,
Lineweaver-Burk plot, etc.

Objectives
After studying this unit, you should be able to:

 derive Michaelis-Menten equation;

 explain the mechanisms of enzyme catalysis;

 draw Lineweaver-Burk plot; and

 describe Km and Vmax. 45

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4.2 ENZYME KINETICS

Kinetic analysis helps to disclose the number and order of the individual steps
involved in the transformation of substrates to products. In the past, data
generated from the experiments of enzyme catalyzed reactions was collected
and analyzed to determine the rate of a reaction. It was found out that at low
concentrations of substrate, the reaction was of first-order with respect to the
substrate. However, at the higher concentrations of substrate, the reaction
became zero-order. Please recall from your chemistry books regarding the
zero order or first order enzyme catalyzed reactions. Generally all single
substrate enzyme catalyzed reactions and even multi-substrate reactions
where concentrations of all but one were kept constant follows the same order.
At constant enzyme concentration, graph of initial velocity [vo] (on y-axis)
against substrate [S] concentration (on x-axis) exhibit a hyperbolic curve
(Fig. 4.1).
Vmax

V0

zero-order
reaction

first-order
reaction

[S0]

Fig.4.1: Graph of initial velocity against substrate concentration for a single

substrate enzyme catalyzed reaction.

The general equation from the graph is

V max [ So ]
vo 
[ So ]  b

Vmax = Maximum velocity = maximum value of vo

b = constant = value of [ So ] where vo = ½ Vmax

In general terms, in a mono substrate enzyme catalyzed reaction and

considering just one substrate binding site per enzyme molecule, substrate [S]
comes in physical contact with enzyme [E] to form an enzyme substrate
complex [ES] complex which eventually undergoes a further reaction and leads
46 to the formation of product [P]. It can be represented as:
Unit 4 Enzyme Kinetics
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rate constant k1 rate constant k2
E+S ES E+P
rate constant k-1

k 1 = rate constant for the association of substrate and enzyme

k 2 = rate constant for the breakdown of enzyme and product

k -1 = rate constant for the dissociation of [ES] complex to form free enzyme
and substrate

The overall rate of reaction is limited by two factors:

1) The amount of concentration of enzyme

2) The breakdown of enzyme-substrate complex

At low substrate concentrations, the overall rate of reaction is limited by the

rate at which enzyme and substrate molecules react to form enzyme-
substrate complex. At constant enzyme concentration the rate of reaction is
proportional to the substrate concentration (first-order reaction). However, at
high substrate concentration enzyme gets saturated with the substrate and
therefore no free enzyme remains available. So the overall rate of reaction
becomes independent of the substrate concentration. The maximum initial
velocity possible is given by the expression.

Vmax = k2 [Eo] Eo = Total enzyme concentration

SAQ 1
The rate of reaction is limited by two factors. Name the factors.

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4.2.1 Michaelis-Menten Equation

Kinetic models used to explain above mentioned findings were proposed by

Michaelis and Menten (1913). The Michaelis-Menten equation demonstrates
the relationship between initial reaction velocity and substrate concentration.
Derivation of this equation begins with the generalized scheme of events
considering a single substrate enzyme catalyzed reaction as stated earlier; 47
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Substrate [S] interacts with enzyme [E] to form an enzyme substrate complex
[ES] complex which eventually breaks down to free enzyme [E] and the
formation of product [P]. The Michaelis and Menten set out the following
scheme as given below:

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

The term k1 denotes the rate constant for the formation of ES complex. ES
complex has two fates, it can dissociate back to enzyme and substrate with
rate constant k-1, or proceed to form product and release the free enzyme with
a rate constant k2. In any enzyme catalyzed reaction, the concentration of
substrate should be five or six orders higher than that of enzyme. The above
model also assumes that k2 << k1. There is every possibility that reaction can
go backward but if we consider only the initial rate of a reaction, we can ignore
the backward reaction.

The overall rate of reaction is termed as initial velocity (v0) and it will depend on
two factors – the rate of formation of product (k2) and the concentration of
enzyme bound with the substrate i.e [ES]

So vo  k 2[ ES ] ………………….equation 1

Michaelis and Menten made two assumptions in their model. First, the
availability of excess substrate [S] >> [E]. Secondly a rapid equilibrium is
established between the reactants ([E] + [S]) and [ES] complex. Moreover, the
breakdown of enzyme-substrate complex is too slow to cause any change in
equilibrium. Hence Michaelis and Menten model is also known as “Rapid
equilibrium model”. Thus at the equilibrium state:

k 1[ E ][ S ]  k  1[ ES ] ...………………… equation 2

[ E ][S ] k  1
  Ks …………………… equation 3
[ ES ] k1

Ks is the dissociation constant. If Eo is the total enzyme concentration, it is

equal to the sum of free enzyme [E] and enzyme bound to the substrate [ES]
as represented in the following equation:

E o  [ E ]  [ ES ] ..…………………..equation 4

E  [ Eo]  [ ES ]

Substituting the value of E in equation 3

([ Eo]  [ ES ])[S ]
 Ks
48 [ ES ]
Unit 4 Enzyme Kinetics
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([ Eo]  [ ES ])[ S ]  Ks[ ES ]

[ Eo][ S ]  [ ES ][ S ]  Ks[ ES ]

[ Eo ][ S ]  Ks[ ES ]  [ ES ][ S ]

[ Eo ][ S ]  [ ES ]( Ks  [ S ])
[ Eo][S ]
[ ES ] 
( Ks  [ S ])

Substituting the value of [ES] in equation 1 we get

[ Eo ][ S ]
So vo  k 2 …………………..equation 5
( K s  [ S ])

Maximum rate of enzyme reaction will be achieved when all the enzyme
molecules are bound to the substrate molecules.

So V max  k 2 [ Eo ] ……………..equation 6

Substituting this value in equation 5 we get

V max[ S ]
vo 
( K s  [ S ])

Michaelis and Menten also made the supposition that initial substrate
concentration [So] is much higher than the initial enzyme concentration [Eo], in
such a scenario the formation of enzyme-substrate complex will have no such
big change in free substrate concentration. The expression for vo will be:

V max[ So ]
vo  ……….equation 7
( K s  [ So ])

The above equation equation 7 is well known as Michaelis-Menten equation

Briggs Haldane modified Michaelis-Menten Plot:

The Michaelis-Menten equation is dependent on the assumption of rapid

equilibrium approach in any enzyme catalyzed reaction. It limits the applicability
of the equation to only rapid kinetic reactions which might not be the case with
many of the other enzyme reactions. Most of enzyme catalyzed reactions
assume a constant concentration of enzyme-substrate complex [ES].

Generally if an enzyme is mixed with high concentration of a substrate, there is

an initial period expressed as pre-steady state (lasts in micro seconds) where
concentration of [ES] slowly builds up. Eventually the concentration of [ES]
builds up and attains a steady state, remains constant over time. The steady-
state concept was introduced by Briggs and Haldane in 1925, a modified
Michaelis-Menten equation. This concept was considered more valid
assumption than the earlier ones. The Michaelis-Menten equation gave
49
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importance to the formation of [ES] while Briggs-Haldane method focuses on
the consistency of [ES] complex, its maintenance at constant concentration
and breakdown to products.

Considering the single substrate enzyme catalyzed reaction one more time. In
this reaction at steady state rate of formation of [ES] will be equal to the rate of
its decomposition to products. Therefore

k 1[ E ][ S ]  k  1[ ES ]  k 2[ ES ] ………………..equation 8

Separating the constant from variables:

[E][S ] k  1  k 2
  Km ………………..equation 9
[ ES] k1

Where Km = Michaelis constant

Substituting Ks with Km in the above equations (4-7)

E  [ Eo]  [ ES ]

Substituting the value of E in equation 9

([ Eo]  [ ES ])[ S ]
 Km
[ ES ]

([ Eo ]  [ ES ])[ S ]  K m [ ES ]

[ Eo ][ S ]  [ ES ][ S ]  K m [ ES ]

[ Eo ][ S ]  K m [ ES ]  [ ES ][ S ]

[ Eo ][ S ]  [ ES ]( K m  [ S ])

[ Eo ][ S ]
[ ES ] 
( K m  [ S ])

Substituting the value of [ES] in equation 1 we get

[ Eo ][ S ]
So vo  k 2 ( K
m  [ S ])

Since V max  k 2 [ Eo ]

Substituting this value in above equation

V max [ S ]
vo 
( K m  [ S ])
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Unit 4 Enzyme Kinetics
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Since substrate concentration is much higher than the enzyme concentration
[S]~ [ So ] so

V max [ So ]
vo  ……………..equation 10
( K m  [ So ])

The above equation equation 10 is similar to well known Michaelis-Menten

equation. The only change is the Km instead of Ks in the denominator. So the
equation retains its previous name of Michaelis-Menten equation and constant
Km is known as Michaelis-Menten constant.

A graph of vo at y-axis vs substrate concentration [S] at x-axis will be in the

form of a hyperbolic curve (Fig. 4.2).

Vmax
Vmax
Reaction velocity (V 0 )

Vmax/2

Km

[S0]

Fig. 4.2: Michaelis-Menten plot of a single substrate catalyzed enzyme reaction

SAQ 2
Explain how the rate of an enzyme-catalyzed reaction reaches a maximum
value at high substrate concentration in a Michaelis-Menten equation?
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4.2.2 Lineweaver Burk Plot

If you look at the Michaelis-Menten plot, you will observe that vo approaches
Vmax in a tangential manner at higher substrate concentrations. So if you want
to determine Vmax and Km from the plot, it will be difficult and unsatisfactory. A
hyperbolic curve nature of the graph makes it difficult to determine the accurate
51
Block 2 Enzyme Kinetics
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value of Vmax and Km. Therefore, to overcome this difficulty, Lineweaver and
Burk (1934) suggested a straight line graph for enzyme catalyzed reactions
obeying Michaelis-Menten equation. They did not made any new assumptions
and derive the Lineweaver-Burk plot, which is also known as double reciprocal
plot. Lineweaver and Burk took the Michaelis-Menten equation and inverted it.
V max [ So ]
vo 
( K m  [ So ])

1 ( K m  [ So ])

vo V max [ So ]

1 [ So ] Km
 
vo V max [ So ] V max [ So ]

1 1 Km
 
vo V max V max[So ]

This equation is known as Lineweaver-Burk equation. It is in the form of

y = mx + c ....... equation 11
which is the equation of a straight line graph. Plot of 1/v against 1/So is linear
and obeys Michaelis-Menten equation (Fig. 4.3). It is known as Lineweaver-
Burk plot or double reciprocal plot.

1 Km
slope =
V0 Vmax

1
 1
Km
Vmax

O 1
S0
Fig. 4.3: Double reciprocal plot of the Michaelis-Menten equation.

4.2.3 Significance of Km and Vmax

Km is also called as Michaelis constant. It is expressed as the substrate

concentration at which velocity is half of Vmax (maximal velocity) (Fig. 4.2). Here
you must note that Km has the units of concentration but it is independent of the
enzyme and substrate concentration. As you know Km is equal to the substrate
concentration at ½ Vmax. And since at Vmax all the enzyme molecules are bound
with substrate to form ES complex, thus the substrate concentration (Km)
required to convert half of the enzyme molecules into ES complex signifies the
affinity of the enzyme for the substrate. When Km value is small, it signifies that
52 enzyme has very high affinity for that substrate i.e. low concentration of
Unit 4 Enzyme Kinetics
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substrate is needed to saturate the enzyme. Similarly, a large value of Km
indicates a relatively high concentration of substrate required to saturate the
enzyme, thus signifying a low affinity of the enzyme for substrate. This is why
Km is also known as affinity constant.

Vmax, the maximal rate represents the rate at which number of substrate
molecules is being converted into product by an enzyme molecule in a unit
time when the enzyme is fully saturated with substrate.

SAQ 3
Do as Directed

a) Michaelis-Menten equation relates the rate of an enzyme-

catalyzed reaction to substrate concentration/product
concentration. (Choose one option)

b) A hyperbolic curve gives an accurate value of Vmax and Km (True/False)

c) Lineweaver-Burk plot is linear/hyperbolic (Choose one option)

d) Km is equal/more to the substrate concentration at ½ Vmax (Choose one option)

4.2.4 kcat and Turnover number

To determine the enzyme efficiency in enzyme kinetics, we are interested to

determine how many maximum molecules of substrate can be converted into
product per catalytic site of a given concentration of enzyme per unit time.

kcat = Vmax/Et

where

kcat = Turnover number,

Vmax = Maximum rate of reaction when enzyme catalytic site is saturated with
substrate Et =Total enzyme concentration or concentration of total enzyme
catalytic sites.

The kcat is a direct measure of catalytic production of product under optimal

conditions. The units of Turn over number (kcat) = (moles of product/sec)/
(moles of enzyme) or sec1.

Metalloenzyme carbonic anhydrase catalyzes interconversion of carbon

dioxide and water to form bicarbonate ions and protons. A turnover number of
400,000 to 600,000 s1 of carbonic anhydrase enzyme suggests that each
enzyme molecule can produce up to 600,000 molecules of product
(bicarbonate ions) per second.
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SAQ 4
Do as Directed:

a) Km is independent of the enzyme and substrate (True/False).

concentration

b) What are the units of turnover number?

c) Kcat is known as turnover number. (True/False)

d) Each enzyme generally catalyzes .................... reaction. (Specific/Any).

4.3 SUMMARY
1) Enzyme kinetics is the basis for enzyme catalyzed biochemical
reactions. Therefore, understanding of enzyme kinetics is important
to understand the biological processes as well as several enzyme
assays being carried out.

2) Kinetics provides a rationale for the complex behavior of enzymes.

The rational is based on simple chemical principles.

3) For single substrate reactions, at constant E, increasing S results

in increased product formation to a point where product formation
no longer increases. This saturation is presumed to reflect the fact
that all E is now in the form of ES.

4) Kinetic model proposed by Michaelis-Menten used equilibrium

assumption for deriving Michaelis-Menten equation.

5) The equilibrium assumption was later modified to introduce a more

valid steady state assumption. The equation remains the same
except the definition of Michaelis constant (Km).

6) The substrate concentration at which velocity is half of Vmax

(maximal velocity) is known as Michaelis constant (Km). This
constant gives an idea about the affinity of the enzyme for its
substrate.

7) The hyperbolic graph of v versus S obtained by Michaelis-Menten

equation proved inadequate for determining the accurate value of
Vmax and Km. Lineweaver and Burk plot provides the solution by
inverting the Michaelis-Menten equation to obtain double reciprocal
54 plot.
Unit 4 Enzyme Kinetics
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4.4 TERMINAL QUESTIONS

1) Why is the rate of an enzyme-catalyzed reaction proportional to
the amount of ES complex?

2) Explain the maximal velocity Vmax in the vo vs S graph?

3) Derive Lineweaver-Burk equation from Michaelis-Menten equation

and give its importance.

4) How a value for Km can be obtained from the vo vs S graph when

vo = 1/2 Vmax?

4.5 ANSWERS
Self-Assessment Questions

1) The rate of reaction is determined by

i) The amount of the concentration of enzyme.
ii) The breakdown of enzyme-substrate complex.

2) At high substrate concentration So, Km <<<< So (numerically), so the

term Km + So in the Michaelis-Menten equation becomes equal to
So. vo = (V So)/So, and So cancels. Therefore, at high So, vo = V .
max max

3) a) Substrate concentration, b) False, c) Linear, d) Equal.

4) a) True, b) sec-1, c) True, d) specific.

Terminal Questions
1) The product formation takes place after ES complex formation in
an enzyme catalyzed reaction [Refer to section 4.2].
2) At high substrate concentrations, enzyme E will be bound to
substrate S. So the maximum amount of E.S is formed under
these conditions. Since the rate is proportional to the amount of
ES, the rate is at a maximum value under these conditions.

3) Refer to section 4.2.2

4) When vo = Vmax/2, then Vmax/2 = Vmax.S/Km + S

cancelling Vmax,

1/2 = S/(K + S)
m

Km + S = 2S

or Km= S at vo = Vmax/2

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4.6 SUGGESTED READINGS

1) David L. Nelson and Michael M. Cox: Lehninger Principles of
Biochemistry6th Ed., W.H. Freeman.

2) Robert K. Murray, Daryl K. Granner, Victor W. Rodwell Harper’s Illustrated

Biochemistry, 27th edition. 2006, McGraw-Hill.

3) Donald J Voetand Judith G. Voet: Principles of Biochemistry 4th ed., John

Wiley and Sons, Inc, USA.

4) Eric E Conn, Paul K Stumpf: Outlines of Biochemistry, John Wiley and

Sons, Inc, USA.

5) S. Shanmugan and T. Sathishkumar: Enzyme Technology, I K International

Publishing House Pvt Ltd, New Delhi.

6) Nicholas C Price and Lewis Stevens: Fundamentals of Enzymology,

Oxford University Press, Oxford, New York, USA.

56
Unit 5

UNIT 5 Bisubstrate Reactions

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BISUBSTRATE REACTIONS

Structure
5.1 Introduction 5.6 Terminal Questions
Objectives 5.7 Answers
5.2 Bisubstrate Reactions 5.8 Suggested Readings
Sequential Reactions
Non-Sequential Reactions

5.3 Alberty Rate Equation

5.4 Differentiating Bisubstrate
Mechanisms
Primary Plots
Non-Steady State Methods
Isotope Exchange

5.5 Summary

5.1 INTRODUCTION
In the previous unit you learnt about the enzyme kinetics of single substrate
enzyme catalyzed reactions. The enzyme catalyzed reactions involving two or
more substrates and yielding two or more products are more common than
the single substrate reaction. Biochemical reactions with two substrates
account for ~60 % of the known biochemical reactions. Therefore it is
necessary to consider the kinetics of “bisubstrate reactions”.

Objectives
After studying this unit, you should be able to:

 determine nomenclature of enzyme catalyzed reactions based on

substrate or products.
 distinguish two different types of bisubstrate kinetic mechanisms;
 write kinetic mechanisms for different types of bisubstrate
reactions using Cleland terminology; and
 explain sequential and ping-pong kinetic mechanisms based on the
patterns observed on double reciprocal (Lineweaver-Burk) plots.
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5.2 BISUBSTRATE REACTIONS

The nomenclature formulated for multisubstrate kinetics is given in the Table 5.1.
Table 5.1: Nomenclature of enzyme catalyzed reactions depending on
substrates or products involved in the reaction.

S.No. Reaction Nomenclature

1. A P Uni uni

2. A+B P Bi uni

3. A+B P1 + P2 Bi bi

4. A+B+C P Ter uni

5. A+B+C P1 + P2 Ter bi

The derivation of velocity equations for multisubstrate reactions is difficult as

compared to single substrate reactions. Kinetic equations for non-rapid
multisubstrate systems can be derived algebraically with the aid of steady state
assumptions. However, as the number of substrates increases the algebraic
manipulations becomes more complicated. In bisubstrate systems, isomerization
of the central complex and subsequent product release steps may be so rapid
that (free enzyme) E, EA (enzyme-substrate A), EAB (enzyme-substrate A and
substrate B complex), EPQ (enzyme - product P- product Q complex) and EQ
(enzyme-product Q) may never attain equilibrium. The kinetic analysis may depend
on the distribution of enzyme in different states as per the rate constants of all the
steps along with the release of products.
Considering the complexity of reactions, we will begin with two substrate two
product reactions taking reactions in a series of steps for example serine
proteases such as chymotrypsin that catalyses the reaction involving two
substrates and yield two products. In the first step, peptide substrate is
hydrolyzed to form two peptide fragment molecules and in the second step, the
water molecule as substrate indirectly supplies the proton and hydroxyl groups
required to complete the hydrolysis. Similarly ATP dependent kinases
catalyses the phosphorylation of protein utilizing ATP as energy molecule and
yielding ADP and phosphoprotein as two product molecules.
Bisubstrate reactions involving two substrate two product (bi-bi) reactions are
generally transfer reactions (including oxidation/reduction reactions) and are
represented as:
AX + B BX + A

Such type of reactions are of two types viz., sequential and non sequential.

5.2.1 Sequential Reactions

In these types of reactions, both the substrates (reactants) bind to the enzyme
before the product is released. If you observe carefully, you will notice that even
in sequential reactions, there could be two ways of reaction mechanisms. It
58 may be ordered or random.
Unit 5 Bisubstrate Reactions
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Ordered Mechanism: In this type of enzyme mechanism, substrate
molecules bind to the enzyme thereby forming a ternary complex and then the
products are released in a defined ordered sequential manner. In other words
the order of binding and leaving the enzyme is compulsory. The order is well
précise and well specified. So this mechanism is also known as Compulsory
order mechanism. Assuming two substrates A and B binds to the enzyme E,
the mechanism is represented as:
+B BX
E + AX E. AX E. AX.B E. A.BX EA E+A

+AX A E + BX
E+B EB E.B.AX E.BX.A E.BX

The diagrammatical summary of the sequences of reactions is also

represented in the form of Cleland plot in which the enzyme is represented as
a horizontal line and the arrows are used to represent the arrival and departure
of substrates and products.

AX B BX A

E EAX EAXB EABX EA E

Some of the well known examples include enzymes such as citrate synthase,
lactate dehydrogenase and aspartate transcarbamoyalase (ATCase). ATP
sensitive K channels and H, K-pumps are also some of the other examples of
compulsory order sequential reactions.

Random Sequential Mechanism: In this type of bisubstrate reactions, there

is no specified order. Any substrate can bind first to the enzyme and any
product may be released first. The formation of ternary complex (enzyme with
two substrates) in this type of mechanism is similar to the ordered
mechanism. The reaction mechanism is represented as:

E + AX E. AX BX EA E+A
+B

E. AX.B E. A. BX
+AX A
E.BX E + BX
E+B EB

In this type of reaction mechanism, there are two separates binding sites on
the enzyme, one for A/AX and other for B/BX.

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The difference between two types of sequential mechanism – Ordered and
Random lies in the fact that enzyme may follow ordered sequential pathway if
first substrate causes a conformational change in the enzyme. However, in
case of random order mechanism there will be separate binding sites on the
enzyme, one for one substrate and another site for another substrate. Random
sequential mechanism is typical in multienzyme complexes such as
hemoglobin, ligand operated channels and tyrosine kinase receptors. The
Cleland plot for such type of reaction mechanism is as follows:
BX A
AX B

EAX EA
E
E. AX.B E. A. BX E
EB E.BX

B AX A BX

SAQ 1
Do as Directed.

a) Chymotrypsin is an example of an enzyme that catalyzes the ..............

type of reactions (single substrate/bisubstrate). (Choose one option)

b) Multisubstrate reactions are more common than the single substrate

reactions. (True/False)

c) Ternary complex is formed in ....................... reactions.

(sequential/non-sequential)

d) Give one example of the enzyme following compulsory order sequential

mechanism.

5.2.2 Non- Sequential Reactions

Ping-Pong Bi-Bi Reactions: These reactions follow non-sequential mechanism
and are also known as double displacement reactions. In such reactions at
least one product gets released from the enzyme before both substrates
bound to the enzyme. A pictorial representation is given below:

AX + E E. AX EX.A EX + A

EX + B EX.B E.BX E + BX

Firstly, AX binds to the enzyme E forming a binary complex. E.AX. The X

represents a small group which on its own cannot participate in the reaction.
The active site of the enzyme is full as this substrate (AX) gets converted to
product on account of intramolecular reorganization in the active site where
60
the bond E-X is formed and the bonding between X-A is broken. The first
Unit 5 Bisubstrate Reactions
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product (A) is also formed which is then released from the enzyme E before
second substrate (B) binds to the enzyme. The second substrate will not bind
free enzyme E but will bind in its modified form E-X. In this type of reaction
mechanism, enzyme has only one binding site since only one substrate binds
at a time. There is no room for the second substrate. The release of first
product exposes the binding site on the enzyme to the second substrate.
Another phase of intermolecular reorganization takes place in the active site of
enzyme where bond B-X is formed and bond E-X breaks down. The release of
second product (BX) follows leaving behind the enzyme in its original form.
Several enzymes such as co-transporters and pumps work as per double
displacement reaction mechanism. Examples include serine proteases such
as trypsin, chymotrypsin and amino transferases.

Cleland plot for ping-pong bi-bi enzyme catalyzed reaction is:

AX A B BX

E E
E.AX EX.A EX EX.B E.BX

SAQ 2
What are two characteristics of an enzyme that catalyzes a reaction
through the ping-pong mechanism?

................................................................................................................

................................................................................................................

................................................................................................................

................................................................................................................

5.3 ALBERTY RATE EQUATION

Bisubstrate enzyme catalyzed reactions observe Michaelis-Menten equation
when one of the two substrates is at constant concentration with respect to the
other substrate. We would like to remind the learner that it will be applicable
only when enzyme has one binding site per substrate or multiple binding sites
per substrate provided there is no interaction between the binding sites. Alberty
derived equation for such reactions is:

=
Vmax [AX𝑜 ][Bo]
vo =
KBm [AX𝑜 ]+ KAX AX B
m [Bo ]+[AX𝑜 ][Bo ]+Ks Km

61
Block 2 Enzyme Kinetics
..........................................................................................................................................................................
Where Vmax is the maximum vo when both the substrates AX and B are saturating
KAXm is concentration of substrate AX which gives ½ Vmax when substrate B is
saturating

KBm is concentration of substrate B which gives ½ Vmax when substrate AX is

saturating KAXs is the dissociation constant for E + AX EAX

The total enzyme concentration is much less than the concentrations of two
substrates. At saturating concentration of [Bo] the Alberty equation will be
o
Vmax [AX𝑜 ]
vo = AX [
Km + AX𝑜 ]

Similarly at the saturating concentration of [AXo] the Alberty equation will be

v o = Vmax [Bo ]
B
Km +[Bo ]

From the above equations, you will observe that two substrate reactions will
also obey Michaelis-Menten equation with respect to one substrate when the
concentration of other substrate is fixed. So, the corresponding Lineweaver-
Burk plot or double reciprocal plot will also be linear.

Several sequential bisubstrate enzyme catalyzed reactions (random order and

compulsory order) will observe Alberty equation when the rate limiting step is
interconversion of the ternary complex (E.AX.B E.A.BX). If you remember,,
the rate limiting step in single substrate reactions is the interconversion of
ES EP.

For double displacement reactions or ping-pong bi-bi enzyme catalyzed non-

sequential reactions, the release of one product from the enzyme in the initial
period of reaction will be irreversible. Hence KAXs = 0 and KAXs. KBm =0. The rate
of reaction will be
=
Vmax [AX𝑜 ][Bo ]
vo =
KB AX
m [AX𝑜 ]+ Km [Bo ]+[AX𝑜 ][Bo ]

5.4 DIFFERENTIATING BI SUBSTRATE

MECHANISMS

5.4.1 Primary Plots

The distinguishment of the bisubstrate enzyme catalyzed reactions can be
made from the primary plots of reaction mechanisms observing Alberty rate
equation. The reaction mechanisms of bisubstrate reaction obeying Alberty
62 rate equation will give linear plots of 1/vo vs 1/[AXo] at constant concentration of
Unit 5 Bisubstrate Reactions
..........................................................................................................................................................................
other substrate [Bo]. Similarly 1/vo vs 1/[Bo] at constant concentration of other
substrate [AXo] will also be a linear plot. The slopes and intercept of these
linear plots includes concentration of the fixed substrate at non-saturating
concentrations. Therefore, the slope as well as intercept of these plots will
change if the experiments are repeated with fixed substrate concentration at a
different value. The linear plots of compulsory or random order sequential
enzyme catalyzed reactions are shown in the Fig. 5.1 a. The double
displacement or Ping-Pong bi bi enzyme catalyzed reaction is also shown in
Fig. 5.1 b. The learner can see from the graph that the linear plot in figures is
independent of the concentration of the fixed substrate. Recall from the last
section, you will notice that KAXs. KBm =0 in ping-pong bi-bi reaction mechanism.
The linear plot of ping-pong bi bi will show several parallel lines if the
experiments are repeated at the different concentrations of the fixed substrate.

1
1
v0 [B0]3
v0
[B0]3 [B0]2
[B0]2 [B0]1

[B0]1

[B0]1 > [B0]2 > [B0]3

[B0]1 > [B0]2 > [B0]3

1 1
[ AX 0 ] [ AX 0 ]

(a) (b)

Fig, 5.1: Graph of plot taken at non-saturating concentrations of both substrates

of the enzyme:
a) for compulsory-order and random –order ternary complex
b) for ping-pong bi-bi reaction mechanisms

The limitation of these plots is that compulsory-order and random order can be
distinguished from the ping pong bi bi reaction mechanism but not from each
other.

5.4.2 Non-steady State Methods-Isotope Exchange

If you recall from your school books regarding radioisotopes, it is one of the
finest technique which can be effectively used to determine enzyme reaction
mechanism. Let us see how? The isotope exchange at chemical equilibrium
helps to determine the reaction mechanism. In ping pong non sequential
mechanism, one of the products is released before the joining of another
substrate to the enzyme. Therefore, exchange of isotope between reactant and
product in a reaction in the absence of other reactants and products suggests
a ping-pong enzyme catalyzed reaction mechanism. Consider the bisubstrate
reaction catalyzed by enzyme sucrose phosphorylase converting
orthophosphate to glucose-1-phosphate. The other substrate in this reaction is
sucrose and other product is fructose. The isotope exchange between
63
Block 2 Enzyme Kinetics
..........................................................................................................................................................................
orthophosphate (substrate) and glucose-1-phosphate (product) in sucrose
catalyzed reaction in the absence of sucrose (other substrate) and fructose
(other product) suggests a ping pond mechanism. Similarly, isotope exchange
between sucrose and fructose in the absence of orthophosphate and glucose-
1-phosphate also suggests a ping pong bi bi enzyme catalyzed reaction
mechanism.

Sucrose Fructose P1 glucose-1-phosphate

E E
E.Sucrose E.Glucose.Fructose E.Glucose E.Glucose.phosphate E.Glucose-1-phosphate

Another useful application of isotopes is in distinguishment of compulsory

order sequential mechanism from random order sequential mechanism. The
perceived change in the rate of equilibrium isotope exchange on account of
changes in the concentration of reactants and products without changing the
concentration of reactants and products will help to determine whether the
bisubstrate enzyme catalyzed reaction mechanism is compulsory or random
order.

The general representation of bisubstrate reaction is:

AX + B BX + A

When the rate of forward reaction is equal to the rate of backward reaction, the
equilibrium will be:

[BX][A]
Keq =
[AX ][B]

The addition of a small amount of radiolabel molecule will not affect the
equilibrium significantly. Therefore, if we add a small amount of radiolabel
molecule B in the reaction and then measure the rate of formation of radiolabel
BX, we can trace the reaction mechanism. In the next step we will increase the
concentrations of A and AX, keeping their ratio [A] : [AX] constant as well as
keeping the equilibrium unchanged. This will lead to change in the rate of
isotope exchange between the reactants and products.

Now consider a compulsory order sequential mechanism:

B AX A BX

E E
EB E.B.AX E.BX.A EBX

AX B BX A

E E
64 EAX E.AX.B E.A.BX EA
Unit 5 Bisubstrate Reactions
..........................................................................................................................................................................
In this bisubstrate reaction, substrate B binds first to the enzyme leading to the
formation of EB. The second substrate AX joins the EB complex to form a
ternary complex E.B.AX. An interchange is followed by the release of first
product A and second product BX in compulsory order. A little increase in the
concentration of one substrate AX and one product A will increase the isotope
exchange between B and BX. The isotope exchange will increase with the
increase in the concentration of A and AX keeping [A] : [AX] constant. You
should see the graph on isotope exchange in Fig. 5.2. If you can recall, there is
formation of ternary complex in compulsory order reactions. Therefore
substantial increase in the concentrations of A and AX will make it difficult for
the substrate B to dissociate from E.B and product BX to dissociate from EBX.
So there will be decrease in the rate of isotope exchange between B and BX.

Let us look again at the compulsory order mechanism with the first substrate
AX binding the enzyme E to form E.AX and then joined by B to form E.AX.B.
An interchange is followed by the release of first product BX and second
product A in a compulsory order. An increase in the concentrations of AX and A
will lead to the free enzyme E forming E.AX and EA complexes. E.AX reacts
with second substrate B and EA doesn’t affect release of BX from E.A.BX.
Therefore the rate of isotope exchange will increase in a hyperbolic manner on
further increase in the concentrations of A and AX. So you will notice a
hyperbolic graph as shown in Fig. 5.3

Isotope
exchange
rate
(B BX)

[AX]

Fig. 5.2: Graph of plot for the isotope exchange from B  BX against [AX] in a
compulsory order bisubstrate enzyme catalyzed reaction when B binds
first.

Isotope
exchange
rate
(B BX)

[AX]

Fig. 5.3: Graph of plot for the isotope exchange from B  BX against [AX] in a
compulsory order bisubstrate enzyme catalysed reaction when AX
binds first.
65
Block 2 Enzyme Kinetics
..........................................................................................................................................................................
Similar results will be obtained in Random order enzyme mechanism.
Rapid Reaction Studies:
Many biochemical processes are too fast for fitting observation using a
standard spectrophotometer. Sometimes these processes can be avoided by
alternative of conditions, other than for enzyme reactions that occur in 0.01 -
100 second timescales, stopped flow methods are helpful. Theorell and
Chance used stopped flow techniques with a double beam detector for
studying enzyme reaction of horse liver alcohol dehydrogenase. The enzyme
catalyzes the formation of formaldehyde from ethanol. The entire reaction is
represented as:

CH3CH2OH + NAD + CH 3CHO + NADH (+H +)

The coenzyme NADH shows absorbance at 350 nm and not at 328nm.

So measuring the differences in the absorbance at 350nm and 328nm will help
to determine the formation of NADH from E.NADH complex (E.NADH

E + NADH). The rate of absorbance at 328 measures the rate of reduction of

NAD + (E.NAD+ E.NADH).

In this type of compulsory order reaction, Chance found out that

interconversion of ternary complex is too fast and the rate limiting step of the
reaction is the release of NADH from E.NADH complex.

SAQ 3
State whether the following statements are True or False

a) The rate equation of Alberty bears resemblance to the

Michaelis-Menten equation (True/False).

b) In the Alberty rate equation for bisubstrate reactions, Vmax denotes

the maximum vo when only one substrate is saturating (True/False).

c) NADH shows absorbance at 350 nm. (True/False)

d) Compulsory-order and random order reaction mechanisms

can be distinguished from ping pong bi bi mechanisms
but not from each other by the primary plots. (True/False)

e) Isotope exchange technique can be used for investigating

reaction mechanisms. (True/False)

5.5 SUMMARY
1) Enzyme kinetics can be complex especially in case of bisubstrate
reactions but can be very informative. Two substrate enzyme reactions
can proceed by several different mechanisms leading to the release of two
66 products.
Unit 5 Bisubstrate Reactions
..........................................................................................................................................................................
2) The bisubstrate reaction mechanism can be sequential (single
displacement reactions) or non-sequential (double displacement reactions
or ping-pong bi-bi).

3) Single displacement reactions can also be either of two subtypes: ordered

sequential or random sequential.

4) The Alberty rate equation for bisubstrate reactions obeys Michaelis-Menten

equation with respect to one substrate at fixed concentration of the other.

5) The kinetic mechanism of bisubstrate reactions can be determined by

steady-state methods and by non-steady state methods such as isotope
exchange and rapid-reaction techniques.

5.6 TERMINAL QUESTIONS

1) What is the difference between random sequential and ordered sequential
reaction mechanisms?

2) Discuss Alberty rate equation?

3) Draw primary plot for distinguishing sequential and non-sequential rate

mechanisms.

4) Discuss rapid-reaction techniques for bisubstrate reactions.

5.7 ANSWERS

Self Assessment Questions

1) a) Bisubstrate, b) True, c) Sequential, d) Lactate Dehydrogenase,

2) Answers:

The characteristics of enzyme catalyzing ping-pong bi bi reaction

mechanism are:

a) A product is formed before the second substrate binds to the

enzyme.

b) The binding of the first substrate causes the enzyme to

change into an intermediate form that will bind the second
substrate.

c) The plot of 1/v vs. 1/[A] as [B] changes will be parallel lines.

3) a) True, b) False, c)True, d) True, e) True

67
Block 2 Enzyme Kinetics
..........................................................................................................................................................................
Terminal Questions
1) Refer to section 5.2.1
2) Refer to section 5.3
3) Refer to section 5.4.1
4) Refer to section 5.4.2 Rapid Reaction Studies

5.8 SUGGESTED READINGS

1) David L. Nelson and Michael M. Cox: Lehninger Principles of
Biochemistry6th Ed., W.H. Freeman.

2) Robert K. Murray, Daryl K. Granner, Victor W. Rodwell Harper’s Illustrated

Biochemistry, 27th edition. 2006, McGraw-Hill.

3) Donald J Voetand Judith G. Voet: Principles of Biochemistry 4th ed., John

Wiley and Sons, Inc, USA.

4) Eric E Conn, Paul K Stumpf: Outlines of Biochemistry, John Wiley and

Sons, Inc, USA.

5) S. Shanmugan and T. Sathish Kumar: Enzyme Technology, I K

International Publishing House Pvt Ltd, New Delhi.

6) Nicholas C Price and Lewis Stevens: Fundamentals of Enzymology,

Oxford University Press, Oxford, New York, USA.

68
Unit 6 Enzyme Inhibition
..........................................................................................................................................................................

UNIT 6
ENZYME INHIBITION

Structure
6.1 Introduction 6.4 Mechanism based inhibitors-
Antibiotics as Inhibitors
Objectives
6.5 Summary
6.2 Enzyme Inhibition-Reversible
and Irreversible 6.6 Terminal Questions

6.3 Reversible Inhibition 6.7 Answers

Competitive Inhibition 6.8 Suggested Readings
Uncompetitive Inhibition
Non-Competitive Inhibition
Mixed Inhibition
Substrate inhibition

6.1 INTRODUCTION
In the previous unit, you have learnt about the kinetics of enzyme catalyzed
reactions. In this unit we will introduce you to important class of molecules
called “enzyme inhibitors”. Enzyme inhibitors are the molecules that regulate
the activity of enzymes in the cell. Inhibitors alter the catalytic action of the
enzyme; as a result slows down the enzyme activity, or in some cases, close
the catalysis. The study of these inhibitors provides wealth of information on
the working of enzymes and their mechanism. The blockage of enzyme
activity can lead to several changes such as correction of metabolic imbalance
or killing of a bacteria or pathogen. Therefore, many of the drug molecules are
enzyme inhibitors and their discovery as well as improvement has been a
major area of research for biochemistry and pharmacology.

Objectives
This unit will give you an overview about different types of enzyme inhibitors.
After studying this unit, you will be able to:
 determine types of enzyme inhibitor;
 find out how inhibitor interacts with the enzyme;
 state the role of inhibitor affecting enzyme kinetic parameters;
 distinguish between reversible and irreversible inhibitors; and
 explain the role of enzyme inhibitors and their classification according
to nature and function. 69
Block 2 Enzyme Kinetics
..........................................................................................................................................................................

6.2 ENZYME INHIBITION-REVERSIBLE

AND IRREVERSIBLE
Molecules that bind to the enzymes and cause a decrease in their activity are
called enzyme inhibitors. These molecules either bind at the active site of
enzyme thereby preventing the substrate molecule to bind to the enzyme or
they may inhibit the catalytic activity of enzyme. You should know that many of
these molecules perform several regulatory roles in the metabolism. Some of
them are used as herbicides or pesticides. Most of the drug molecules also
act as enzyme inhibitors. Natural enzymes inhibitors e.g. poison are a part of
the defense mechanisms in wild life animals.

Enzyme inhibitions are mainly classified into two types:

a) Reversible Inhibition

b) Irreversible Inhibition

6.3 REVERSIBLE INHIBITION

In this unit, we will discuss about the inhibition of simple single substrate
enzyme catalyzed reactions. In reversible enzyme inhibition, loss of the
enzyme activity due to inhibitory molecule is reversible. Enzyme activity gets
restored on the removal of inhibitor. These inhibitors bind non-covalently and
give rise to different kinds of inhibition. Multiple weak bonds between the
inhibitor and the enzyme combine to give strong binding which prevents the
formation of product. They can be easily removed by dilution or dialysis to
restore full enzyme activity. Reversible inhibitors tend to form equilibrium with
an enzyme leading to certain level of inhibition.

6.3.1 Competitive Inhibition

There is a direct competition between the substrate and the inhibitor for the
binding site of enzyme in a competitive inhibition. If inhibitory molecule is bound
to the enzyme then substrate will not be able to bind it and vice versa. In such
kind of scenario, inhibition can be overcome by increasing substrate
concentration in comparison to inhibitor. Therefore, the maximum velocity
(Vmax) of the reaction will remain unchanged while Km will be decreased in a
competitive inhibition. Most of the competitive inhibitors are structurally similar
to the substrate molecules.
k1 k2
E+S ES E+P
k-1
I + I

EI
[𝐸][𝐼]
The dissociation constant (Ki) or inhibitor constant for the reaction is
[𝐸𝐼] = 𝐾𝑖 𝑜𝑟
[𝐸 ][𝐼] [𝐸][𝐼]
= 𝐾𝑖 𝑜𝑟 = [𝐸𝐼] ……… equation 1
70 [𝐸𝐼] [𝐾𝑖 ]
= [𝐸𝐼 ]
= 𝐾𝑖 𝑜𝑟
Unit 6 Enzyme Inhibition
..........................................................................................................................................................................
I = Inhibitor
EI = Enzyme bound to Inhibitor
E = Free Enzyme
Eo = Total enzyme
ES = Enzyme bound to substrate
As you know in the previous unit, using steady state assumption, Michaelis-
Menten constant for the rate of reaction is given by the following equation.

[ E ][S ] k  1  k 2
  Km ……………. equation 2
[ ES ] k1

Eo  [ E ]  [ ES ]  [ EI ] ……………. equation 3

Substituting the value of [EI] as given in equation 1

[ E ][ I ]
Eo  [ E ]  [ ES ] 
Ki
[I ]
Eo  [ E ](1  )  [ ES ]
Ki

[ Eo]  [ ES ]
E
[I ]
(1  )
Ki

Substituting the value of [E] in equation 2:

([ Eo]  [ ES ])[S ]
 Km
I
(1  [ ])[ ES ]
Ki

([ Eo][S ]) I
 [ S ]  Km(1  [ ])
[ ES ] Ki

([ Eo][S ]) I
 Km(1  [ ])  [ S ]
[ ES ] Ki

([ Eo][ S ])
 [ ES ]
I
Km (1  [ ])  [ S ]
Ki
vo  k 2[ ES ] ……………. equation 4

(Ref: Michaelis Menten equation in the previous Unit-4, Block-2)

Putting the value of [ES] in equation 4
k 2[ Eo][ S ]
vo 
I
Km (1  [ ])  [ S ] 71
Ki
Block 2 Enzyme Kinetics
..........................................................................................................................................................................
Vmax = k2 [E0] ……………. equation 5
(Ref: Michaelis Menten equation in the previous Unit-4, Block-2)

V max[ S ]
vo 
I
Km(1  [ ])  [ S ]
Ki

Since inhibitor concentration is generally of the same order of magnitude as

the substrate concentration and much higher than the enzyme concentration [I]
~ [ Io ] just as [S] ~ [ So ]. The above equation can be expressed as

V max[ So]
vo 
Io ……………. equation 6
Km (1  [ ])  [ So]
Ki

This equation is similar to Michaelis- Menten equation and the Km is increased

by a factor

Io Io
Km(1  [ ]) or K’m = Km(1  [ ])
Ki Ki

The Lineweaver- Burk equation and plot (Fig. 6.1) of competitive inhibition will
be:
1 K'm 1
 
vo V max[ So] V max

1
+ Competitive
v0 [I0]3
inhibitor
1 Km [I ]
Slope = 1+ 0 [I0]2
v0 Vmax Ki
[I0]1
Uninhibited

1 Km
K Slope =
m Vmax
1
[I0]3 > [I0]2 > [I0]1
Vmax

1 1 1
 [S0 ] [S 0 ]
[I0 ]
K m 1+
Ki
(a) (b)

Fig,6.1(a): Lineweaver-Burk plot for competitive inhibitor (b) Plot at fixed

enzyme concentration but different inhibitor concentrations

Examples of Competitive Inhibition: Succinate dehydrogenase (SDH) enzyme

72 converts succinate to fumarate. Succinate is competitively inhibited by
Unit 6 Enzyme Inhibition
..........................................................................................................................................................................
malonate. Malonate (CO2.CH2.CO2) is structurally similar to fumarate having
two carboxyl groups. Similarly, inhibition of xanthine oxidase enzyme by
allopurinol leads to decreased formation of uric acid and act as a remedy for
gout.

CO2.CH2.CH2.CO2 CO2.CH CH.CO2

Succinate Fumarate

SAQ 1
Draw Lineweaver- Burk plot for different inhibitory concentrations of a
competitive inhibitor at the fixed enzyme concentrations.

................................................................................................................

................................................................................................................

................................................................................................................

................................................................................................................

6.3.2 Un-Competitive Inhibition

Uncompetitive inhibitors are those inhibitors who do not compete with the
substrate and do not bind to the free enzyme. They bind to the enzyme
substrate complex thereby forming a dead end complex. It is quite likely that
binding of substrate to the enzyme may cause some conformational changes
and reveals an inhibitor binding site where inhibitor can bind. This type of
inhibition cannot be overcome by excess of substrate. Both Km and Vmax gets
changed in this type of inhibition pattern.

k1 k2
E+S ES E+P
k-1
I + I
ESI

The inhibitor constant Ki is as given below:

[ ES ][ I ] [ ES ][ I ]
 Ki or  [ ESI ] ..............................equation 7
[ ESI ] Ki

I = Inhibitor

ESI = Enzyme bound to Inhibitor and substrate

E = Free Enzyme

ES = Enzyme bound to substrate 73

Block 2 Enzyme Kinetics
..........................................................................................................................................................................

Eo = Total enzyme

Using steady state assumption, Michaelis- Menten constant for the rate of
reaction is given by

[ E ][S ] k  1  k 2
  Km ……………. equation 2
[ ES ] k1

Eo  [ E ]  [ ES ]  [ ESI ] ……………. equation 8

Substituting the value of [ESI] as given in equation 7

[ ES ][ I ]
Eo  [ E ]  [ ES ] 
Ki

[I ]
Eo  [ E ]  [ ES ](1  )
Ki

[I ]
E  [ Eo ]  [ ES ](1  )
Ki

Substituting the value of E in equation 2

[I ]
[ Eo ]  [ ES ](1  )][ S ]
Ki
 Km
[ ES ]

[ Eo ][ S ] I
 (1  )[ S ]  Km
[ ES ] Ki

[ Eo][ S ] I
 Km  (1  )[ S ]
[ ES ] Ki

[ Eo ][ S ]
 [ ES ]
I
Km  (1  )[ S ]
Ki

vo  k [ES]
2
……………. equation 4

(Ref: Michaelis Menten equation section in the previous Unit 4, Block 2)

Putting the value of [ES] in equation 4

[ Eo ][ S ]
vo  k 2
I
Km  (1  )[ S ]
Ki
74
Unit 6 Enzyme Inhibition
..........................................................................................................................................................................
Vmax = k2[E0] ……………. equation 5

(Ref: Michaelis Menten equation section in the previous Unit-4, Block-2)

Since inhibitor concentration is generally of the same order of magnitude as

the substrate concentration and much higher than the enzyme concentration [I]
~ [ Io ] just as [S] ~ [ So ]. Thus continuing similarly, above equation can be
expressed as

V max[ So]
vo 
Io ……………. equation 9
Km  (1  )[ So]
Ki

[ Io]
Dividing the numerator and denominator by (1  ) gives
Ki

V max[ So]
Io
(1  )
vo  Ki
Km
 [ So ]
Io
(1  )
Ki

The above equation is similar to Michaelis-Menten equation. However, the

constants Km and Vmax are changed as following:

V max
V  max 
Io
(1  )
Ki
and

Km
K m 
Io
(1  )
Ki
The Lineweaver-Burk equation in the presence of an uncompetitive inhibitor is:

1 K'm 1
 
vo V  max[ So] V max
The slope of a Lineweaver-Burk plot will remain unchanged as given by the
following equation:

Km
[ Io]
(1 
K m Ki Km
 
V  max V max V max
[ Io]
(1 
Ki
75
Block 2 Enzyme Kinetics
..........................................................................................................................................................................
The x-intercept as well as y-intercept of the Lineweaver-Burk plot in the
presence of an uncompetitive inhibitor (Fig. 6.2) will change but the slope will
remain unaltered.
1
[I0]3
+ uncompetitive v0
[I0]2
1 inhibitor
v0 Km [I0]1
1 Slope =
[I ] Vmax
Vmax 1+ 0
Ki
Uninhibited

Km
Slope =
Vmax
1
Vmax [I0]3 > [I0]2 > [I0]1

1 1
[I ] 1 [S 0 ] [S 0 ]
 1 1+ 0
Km Ki Km

(a) (b)

Fig.6.2 (a): Lineweaver-Burk plot of enzyme inhibited by an uncompetitive

inhibitor (b) Plot at fixed enzyme concentration but different inhibitor
concentrations.

Example of Uncompetitive Inhibitor: Enzyme aryl sulphatase is uncompetitively

inhibited by hydrazine. Generally uncompetitive inhibition pattern is not seen in
single substrate reactions but several bi-substrate reactions shows this kind of
pattern.

SAQ 2
Draw Lineweaver- Burk plot for different inhibitory concentrations of an
uncompetitive inhibitor at the fixed enzyme concentrations.

.......................................................................................................................

.......................................................................................................................

.......................................................................................................................

.......................................................................................................................

6.3.3 Non-Competitive Inhibition

Non-competitive inhibitory molecules react with the enzyme at a site other than
the active site. The inhibitor can bind to the free enzyme or when it is bounded
to the substrate molecule. It destroys the catalytic activity of the enzyme. The
impact of inhibitor can not be overcome by increasing the substrate
concentration. Apparently, Km value will not be changed but Vmax will change. Let
76 us consider a simple situation involving single substrate reaction
Unit 6 Enzyme Inhibition
..........................................................................................................................................................................
+S
E+S ES E+P
-S
I +I I +I
+S
EI ESI
-S
Since ES can be formed via two alternative routes and considering the fact that
inhibitory rates could vary in binding the free enzyme or when it is bound to the
substrate the situation will become quite complex. In order to simplify the
above model, we assume that substrate binding will have no effect on the
binding of inhibitor. The dissociation constant (inhibitor constant) for both the
reactions E + I EI and ES + I ESI will then be the same. The total
enzyme concentration will be reduced in the presence of inhibitor and it will
lead to decrease in Vmax. The value of Km will not change because binding of
inhibitor does not affect the binding of substrate to the enzyme or vice versa.

In the presence of non-competitive inhibitor

[ E ][ I ] [ ES ][ I ]
 Ki  ……………… equation 10
[ EI ] [ ESI ]

Eo  [ E ]  [ ES ]  [ EI ]  [ ESI ] ……………… equation 11

Substituting the value of [EI] and [ESI] as given in equation 10

[ E ][ I ] [ ES ][ I ]
Eo  [ E ]  [ ES ]  
Ki Ki

[ E ][ I ] [ ES ][ I ]
Eo  [ E ]   [ ES ] 
Ki Ki

[I ] [I ]
Eo  [ E ](1  )  [ ES ](1  )
Ki Ki

[I ]
Eo  ([ E ]  [ ES ])(1  )
Ki

[ Eo ]
E  [ ES ]
[I ]
(1  )
Ki

Substituting the value of E in equation 2

[ Eo ]
(  [ ES ])[S ]
[I ]
(1  )
Ki  Km
[ ES ]

77
Block 2 Enzyme Kinetics
..........................................................................................................................................................................

[ Eo][S ]
(  [ ES ][S ])
[I ]
(1  )
Ki  Km
[ ES ]

[ Eo][ S ] [I ]
 [ ES ][S ](1  )
[I ] Ki
(1  )
Ki  Km
[ ES ]

[ Eo][S ] [I ] [I ]
 [ S ](1  )  Km(1  )
[ ES ] Ki Ki

[ Eo][S ] [I ] [I ]
 Km(1  )  [ S ]( I  )
[ ES ] Ki Ki

[ Eo][S ] [I ]
 ( Km  S )( I  )
[ ES ] Ki

[ Eo][ S ]
 [ ES ]
[I ]
( Km  [ S ])(1  )
Ki

vo  k [ES]
2
……………. equation 4

(Ref: Michaelis Menten equation section in the previous Unit-4, Block-2)

Putting the value of [ES] in equation 4

[ Eo][S ]
vo  k 2
[I ]
( Km  [ S ])(1  )
Ki

V max  k 2[ Eo] ……………. equation 5

(Ref: Michaelis Menten equation section in the previous Unit-4, Block-2)

Continuing similarly as in previous sections,

V max[ So]
vo 
[ Io] ……………. equation 12
( Km  [ So])(1  )
Ki
This is similar to the Michaelis-Menten equation with Vmax divided by a factor
[ Io]
(1  )
Ki
78
Unit 6 Enzyme Inhibition
..........................................................................................................................................................................

Vmax
V'max =
 I0 
1+ 
 Ki 
The Lineweaver-Burk equation in the presence of a noncompetitive inhibitor is:

1 Km 1
 
vo V  max[ So] V  max

The Lineweaver-Burk plot (Fig. 6.3) of a non-competitive inhibitor is as given

below:
1
+ non-competitive v0
inhibitor [I0]3
1 [I0]2
K [I ]
v0 Slope = m 1+ 0
Vmax Ki [I0]1
Uninhibited
1 [I0 ]
1+
Vmax Ki
Km
Slope =
Vmax
1
K
m 1 [I0]3 > [I0]2 > [I0]1
Vmax

1 1
[ S0 ] [S 0 ]

(a) (b)

Fig, 6.3 (a): Lineweaver-Burk plot for non-competitive inhibitor (b) Plot at fixed
enzyme concentration but different inhibitor concentrations.

Example of Noncompetitive Inhibition: Chymotrypsin is noncompetitively

inhibited by H+. Heavy metal ions and small organic molecules bound to the –
SH groups of the cysteine moiety in an enzyme can also lead to the
noncompetitive inhibition.

SAQ 3
Draw Lineweaver- Burk plot for different inhibitory concentrations of a
noncompetitive inhibitor at the fixed enzyme concentrations.

.......................................................................................................................

.......................................................................................................................

.......................................................................................................................

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79
Block 2 Enzyme Kinetics
..........................................................................................................................................................................
6.3.4 Mixed Inhibition
Mixed inhibition occurs when an inhibitor may binds to the enzyme as well as
enzyme substrate complex. However, the affinity with which inhibitor binds to
two different states may varies. It may bind with greater affinity when the
enzyme is free than the enzyme substrate complex or vice versa. The
inhibition is a mixture of competitive inhibition and uncompetitive inhibition. If the
inhibitor has equal affinity for both the states of enzyme (free as well as bound
to the substrate), mixed inhibition will become non-competitive inhibition. In
mixed inhibition, inhibitor binds to the enzyme at a site different from the active
site, the site where substrate binds. As you know, there are two processes by
which inhibitor can bind to the enzyme.

E+I EI (inhibitor constant Ki)

and

ES + I ESI (inhibitor constant K1)

Therefore

[ E ][ I ]
 Ki
[ EI ]
and
[ ES ][ I ]
K1 
[ ESI ]

As you know in a single substrate reaction,

[ E ][S ]
Km 
[ ES ]

Eo  [ E ]  [ ES ]  [ EI ]  [ ESI ] …………………………..equation 13

Since Ki and K1 are not similar, they are different. So

[ E ][ I ] [ ES ][ I ]
Eo  [ E ]  [ ES ]  
Ki K1

[ E ][ I ] [ ES ][ I ]
Eo  [ E ]   [ ES ] 
Ki K1

[I ] [I ]
Eo  [ E ](1  )  [ ES ](1  )
Ki K1

 
[E 0 ]  [ES] 1  [I] 
 K I   [E]
 [I] 
1  
80  Ki 
Unit 6 Enzyme Inhibition
..........................................................................................................................................................................
Substituting for [E] in equation 2, the expression for Km :

  
[E 0 ]  [ES] 1  [I]   [S ]
 K I  
 Km
 [I] 
1   [ES]
 K i 

 [I]   [I] 
[E 0 ][S] - [S] [ES] 1    K m [ES]1  
 KI   Ki 

  [I]   [I]  
[ES]  [S]  1    K m 1  
  K    [E 0 ][S]
  KI   i  

[E 0 ][S]
 [ES] 
 [I]   [I ] 
[S] 1    K m 1  
 KI   Ki 
vo  k 2[ ES ] ……………. equation 4

(Ref: Michaelis Menten equation section in the previous Unit-4, Block-2 of

BBCCT-107)

Putting the value of [ES] in equation 4

k 2 [ E 0 ][ S ]
v0 
 [I]   [I ] 
[S] 1    K m 1  
 KI   Ki 

V max  k 2[ Eo] ……………. equation 5

(Ref: Michaelis Menten equation section in the previous Unit-4, Block-2 of

BBCCT-107) Continuing similarly as in previous sections:

Vmax [ So]
v0 
 [I ]   [I ] 
[S] 1  0   K m 1  0  ……………. equation 14
 KI   Ki 
Dividing the numerator and denominator by (1 + (I0]/Ki)),

Vmax
[ So ]
 [I 0 ] 
1  
 K I 
v0 
 [I ] 
K m 1  0 
 Ki 
[So] 
 [I 0 ] 
1  
 KI 
81
Block 2 Enzyme Kinetics
..........................................................................................................................................................................
The above equation assumes the same form as the Michaelis-Menten equation
and can be written as:
'
Vmax [ So ]
v0 
[ So]  K m'
where

 [I0 ] 
1  
Vmax  K i 
'
Vmax  and K m  K m
'

 [I0 ]   [I0 ] 
1   1  
 KI   K I 
The Lineweaver-Burk equation will be:
1 K' 1 1
 'm  '
v0 Vmax [ So] Vmax

From the equation, it appears that the Lineweaver-Burk plot will be linear.
However, Km, Vmax and slope will be affected by the inhibitor. The Lineweaver-
Burk plot will be represented as shown in the Fig. 6.4.
+ inhibitor 1
v0
1 Km [I ]
Slope = 1+ 0
v0 Vmax Ki + inhibitor
1 [I ] Km [I ]
1+ 0 Slope = 1+ 0
Uninhibited Vmax Ki Vmax Ki
1 [I ] Uninhibited
1+ 0
Vmax Ki [I ]
Km 1+ 0
Ki Km
Slope = 1 Slope =
1 Vmax  Vmax
 Km 1
Km [I0 ]
1+ 1
Vmax Ki
Vmax

[I0 ] 1
1+
Ki [S0 ]
1 1 1
 
Km [I ] Km [S 0 ]
1+ 0
Ki
(a) (b)
Fig, 6.4 : Lineweaver-Burk plots showing the effect of mixed
inhibition: (a) K1>Ki; (b) K1<Ki.

6.3.5 Substrate Inhibition

In enzyme catalyzed reactions, when the amount of enzyme is kept constant
and substrate concentration is increased gradually the rate of reaction will
increase and the velocity curve will reach a maximum. A further increase in
substrate concentration does not increase the rate of reaction and the velocity
curve falls. This event occurs on account of substrate inhibition whenever a
dead-end-enzyme substrate complex is formed thereby inhibiting its own
conversion to the product. Several enzymes are inhibited by their own
substrates. For example, enzyme succinate dehydrogenase catalyzes
dehydrogenation of substrate succinate. If you will look at the structure of
82 succinate, you will see two carboxyl groups at the two ends of the molecule.
Unit 6 Enzyme Inhibition
..........................................................................................................................................................................
The enzyme reaction takes place when both the carboxyl groups of the
substrate bind to the enzyme. Excess concentration of succinate will lead to
an increased possibility of carboxyl groups from two different molecules of
succinate binding to the same enzyme, thereby stopping the enzyme reaction
due to the formation of a dead-end complex. The graphs of characteristic
substrate inhibition are shown in the Fig. 6.5a and 6.5b.
1
v0
V0

1
 1
Km
Vmax

[S0 ] 1
[ S0 ]
a) Michaelis-Menten plot b) Lineweaver-Burk plot

Fig. 6.5: Graphs on Substrate inhibition

The biological significance of substrate inhibition of phosphofructokinase

ensures resources are not dedicated to the formation of ATP when it is in
plenty. Another enzyme DNA metyltransferases catalyze transfer of methyl
group to DNA. Their substrate inhibition leads to faithfully copy of DNA
methylation patterns when cells divide while preventing de novo methylation of
methyl-free promoter regions.

Regarding the enzyme kinetics, if you recall from the uncompetitive inhibition

V max[ So]
Io
(1  )
vo  Ki
Km
 [ So ]
Io
(1  )
Ki

If the inhibitor is identical to the substrate, the equation will become:

Vmax
[ So ]
 [ So ] 
1  
 K i  Vmax [ So]
v0  
Km  [ So ] 
[So]  [ So] 1    Km
 [ So ]   K i 
1  
 K i 

83
Block 2 Enzyme Kinetics
..........................................................................................................................................................................
When the substrate concentration is low [S] the term [S]/Ki becomes negligible
and the equation will be the normal Michaelis-Menten equation. However, when
the substrate concentration is high, then

 [ So ]   [ So ] 
[ So] 1  K  + Km ~ [ S ] 1  
 o  K 
 i   i 

The rate of equation will be

Vmax
v0 
[ So ] …………………………..equation 15
1
Ki

As shown in the above equation, vo will decrease on an increase of substrate

concentration due to substrate inhibition.

SAQ 4
Do as Directed:
a) Mixed inhibition is a mixture of competitive inhibition and
uncompetitive inhibition. (True/False)
b) If the inhibitor has equal affinity for both the states of enzyme
(free as well as bound to the substrate), mixed inhibition will
become non-competitive inhibition. (True/False)
c) The substrate inhibition of phosphofructokinase ensures
resources are not dedicated to the formation of ATP when
it is in less/excess. (Pick one option)
d) Succinate has one/two carboxyl groups. (Pick one option)

6.4 MECHANISM BASED INHIBITORS-

ANTIBIOTICS AS INHIBITORS
Irreversible Enzyme Inhibition
Irreversible inhibition is different from temporary enzyme inactivation by the
reversible inhibitor. The enzyme activity is lost on the binding of inhibitor
molecule to enzyme and its activity cannot be recovered afterwards. These
inhibitory molecules are highly specific and can modify enzyme 3D structure.
The enzyme gets inactive or there is time dependent loss of enzyme
concentration. Inhibition cannot be removed by dilution or dialysis without losing
enzyme activity. Inhibitors generally form or break covalent bonds with the
amino acid residues essential for substrate binding, catalysis or maintenance
of enzyme conformation.
Examples: Heavy metal ions such as mercury, lead, aldehydes and
haloalkanes. Alkylating agents such as iodoacetate and iodoacetamide forms
covalent linkages with –SH groups of the enzyme.
84
Unit 6 Enzyme Inhibition
..........................................................................................................................................................................
 
E-SH + ICH2.CO 2
E-S-CH2.CO 2
+ HI
The mechanism-based inhibitors are modified substrates that are also
known as suicide inhibitors or suicide inactivators. They modify the active site
of enzyme irreversibly. These suicide inactivators or inhibitors are generally
inactive unless they bound to the enzyme at its active site. The inhibitor binds
to the enzyme as substrate and is catalyzed in a similar manner. The
mechanism of catalysis generates a chemically reactive intermediate that
leads to the inactivation of enzyme by covalent modification. These inactivators
are used to unravel the mechanism of enzyme reaction and substrate
reactivity. This mechanism has proved to be a boon for rational drug designing
by pharmaceutical companies to determine the molecules which could be
synthesized by the chemists as pharmaceutical agents.
Several important drugs are well known examples of irreversible inhibitors.
Widely used drugs such as penicillin act by covalently inhibiting the enzyme
transpeptidase, thereby preventing the synthesis of bacterial cell walls and
thus killing the bacteria. You must have heard of tablet aspirin. The drug aspirin
inhibits the enzyme cyclooxygenase thereby reducing the synthesis of
inflammatory signals. Enzyme monoamine oxidase deaminates
neurotransmitters such as dopamine and serotonin, and lowers the levels of
these hormones in the brain. A neurodegenerative disease such as
Parkinson’s disease is linked with low levels of dopamine, while depression is
related with low levels of serotonin. Suicide inhibitor such as drug (-)deprenyl,
is widely used to treat Parkinson disease and depression.

6.5 SUMMARY
1) Enzyme inhibition can be either reversible or irreversible. The different
modes of reversible enzyme inhibition can be distinguished by their effects
on the kinetic behavior of enzymes: competitive, uncompetitive or non-
competitive.
2) Competitive inhibitors compete with the substrates for the same binding
site available on the enzyme. Therefore the graphs of these inhibitors
show that Km is increased but Vmax remain unchanged in the presence of
an inhibitor.
3) The uncompetitive inhibitor does not compete with the substrate.
However, it binds at a site other than the substrate binding site on the
enzyme-substrate complex. The graphs of these inhibitors show that Km
and Vmax is altered in the presence of an inhibitor but the slope remains
unchanged.
4) Non-competitive inhibitors also bind at a different site other than the
substrate binding site on the enzyme and enzyme substrate complex. In
the presence of such inhibitors, Km remains unchanged, however Vmax is
decreased.
5) Mixed inhibition is a mixture of competitive inhibition and uncompetitive
inhibition.
6) Substrate inhibition seems to be a type of uncompetitive inhibition, the
extra substrate molecule acts as an inhibitor. 85
Block 2 Enzyme Kinetics
..........................................................................................................................................................................
7) Irreversible inhibitors can be used to map the active site of enzyme. They
bind to the active site of enzyme and modify it covalently and hence
cannot dissociate from the enzyme. They reduce the concentration of
enzyme present.
8) Mechanism based inhibitors or suicide inhibitors are processed by the
enzyme in a catalytic mechanism resulting in the formation of a reactive
compound that inactivates or inhibits the enzyme. These inhibitors are
widely used for rational drug designing.

6.6 TERMINAL QUESTIONS

1) Discuss uncompetitive inhibition and derive the equation for an
uncompetitive inhibitor.
2) Distinguish between reversible and irreversible inhibition?
3) Explain the substrate inhibition.
4) What are the mechanism-based inhibitors? How they help in drug designing?

6.7 ANSWERS
Self-Assessment Questions
1) Refer to Fig. 6.1 b
2) Refer to Fig. 6.2 b
3) Refer to Fig. 6.3 b
4) a) True, b) True, c) Excess, d) Two

Terminal Questions
1. Refer to section 6.3.2
2. Refer to section 6.2
3. Refer to section 6.3.5
4. Refer to section 6.4

6.8 SUGGESTED READINGS

1) David L. Nelson and Michael M. Cox: Lehninger Principles of Biochemistry
6th Ed., W.H. Freeman.
2) Robert K. Murray, Daryl K. Granner, Victor W. Rodwell Harper’s Illustrated
Biochemistry, 27th edition. 2006, McGraw-Hill.
3) Donald J Voet and Judith G. Voet:Principles of Biochemistry4th ed., John
Wiley and Sons, Inc, USA.
4) Eric E Conn, Paul K Stumpf: Outlines of Biochemistry, John Wiley and
Sons, Inc, USA.
5) S. Shanmugan and T. Sathish Kumar: Enzyme Technology, I K
International Publishing House Pvt Ltd, New Delhi.
6) Nicholas C Price and Lewis Stevens: Fundamentals of Enzymology,
86 Oxford University Press, Oxford, New York, USA.