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
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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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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
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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
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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.

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