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Enzyme Kinetics

The document discusses enzyme kinetics and the Michaelis-Menten model of enzyme-catalyzed reactions. It describes how the rate of product formation (V) varies with substrate concentration and derives the Michaelis-Menten equation. Key terms are defined, including KM (Michaelis constant) and Vmax (maximum reaction rate). Different types of reversible enzyme inhibition are also summarized, including their effects on KM and Vmax in Lineweaver-Burk plots.

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Lyra Lasangre
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896 views26 pages

Enzyme Kinetics

The document discusses enzyme kinetics and the Michaelis-Menten model of enzyme-catalyzed reactions. It describes how the rate of product formation (V) varies with substrate concentration and derives the Michaelis-Menten equation. Key terms are defined, including KM (Michaelis constant) and Vmax (maximum reaction rate). Different types of reversible enzyme inhibition are also summarized, including their effects on KM and Vmax in Lineweaver-Burk plots.

Uploaded by

Lyra Lasangre
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
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Enzyme Kinetics

Rate of Enzyme Catalysis vs Substrate


Concentration
For many enzymes, the rate of
catalysis, V, varies with the
substrate concentration, [S].
V is defined as the number of
moles of product formed per
second.
At a fixed concentration of
enzyme, V is almost linearly
proportional to the initial
increasing concentration of [S].
At very high [S], V is
independent of [S].

The Michaelis-Menten Approach to Enzyme


Kinetics
(Leonor) Michaelis and (Maud) Menten devised a model for the kinetics of
enzyme-catalyzed reactions.
The model proposed is the one that accounts for the kinetic properties of many
enzymes:

An enzyme, E, combines with S to form an ES complex, with a rate constant k1.


The ES complex has two possible fates. It can dissociate to E and S, with a rate
constant of k-1, or it can proceed to form product, P, with a rate constant k2.
Michaelis and Menten made a derivation of these kinetic characteristics to arrive
at the Michaelis-Menten Equation:

Vinit =

Vmax [S]
KM + [S]

Michaelis-Menten
equation

Michaelis-Menten Equation Derivation


For an enzyme-catalyzed reaction
E + S

k1
k-1

ES

k2

The rates of formation and breakdown of ES are


given by these equations
rate of formation of ES = k 1 [E][S]
rate of breakdown of ES = k -1 [ES] + k2[ES]

At the steady state


k1 [E][S] = k -1[ES] + k 2[ES]

Michaelis-Menten Equation Derivation

(cont.)

When the steady state is reached, the concentration


of free enzyme is the total less that bound in ES
[E] = [E] T - [ES]

Substituting for the concentration of free enzyme and


collecting all rate constants in one term gives
([E]T - [ES]) [S]
[ES]

k-1 + k2
k1

= KM

Where KM is called the Michaelis constant

Michaelis-Menten Equation Derivation

(cont.)

It is now possible to solve for the concentration of the


enzyme-substrate complex, [ES]
[E]T [S] - [ES][S]
= KM
[ES]
[E]T [S] - [ES][S] = KM[ES]
[E]T [S] = [ES](K M + [S])

Or alternatively

[ES]

[E] T [S]
=
KM + [S]

Michaelis-Menten Equation Derivation

(cont.)

In the initial stages, formation of product depends only on the


rate of breakdown of ES

Vinit = k2 [ES]

k 2[E]T [S]
=
KM + [S]

If substrate concentration is so large that the enzyme is


saturated with substrate [ES] = [E]T

Vinit = Vmax = k2 [E]T


Substituting k2[E]T = Vmax into the top equation gives
Vinit =

Vmax [S]
KM + [S]

Michaelis-Menten
equation

The Meaning of KM or the Michaelis constant


Initial concentration of substrate at Vmax or at half
saturation of E with S
Ratio of the constants for the breakdown of ES to
constants of its formation:
[E]T [S] - [ES][S]
[ES]

= KM

[E]Taffinity
[S] - [ES][S]
= KS:
M[ES]
of E for
if k2 is <k-1 or k1,
A measure of the
neglecting k2, therefore[E]
KMT [S]
= k=-1/k
1
[ES](K
M + [S])

The lower the KM value, the more affinity has the


enzyme for its substrate

The Meaning of KM or the Michaelis constant


When [S]= KM, the equation reduces to

V=

Vmax [S]
KM + [S]

Vmax [S]
[S] + [S]

Vmax
2

Linearizing The Michaelis-Menten Equation


o It is difficult to determine Vmax experimentally
o The equation of Michaelis and Menten is for a hyperbola

Vmax [S]
V=
KM + [S]

(an equation for a hyperbola)

o The equation can be transformed into the equation for a


straight line by taking the reciprocal of each side

1 =
V

KM + [S]
Vmax [S]

KM

Vmax [S]

KM
1 =
+ 1
V
Vmax [S]
Vmax

[S]
Vmax [S]

Lineweaver-Burk Plot
The Lineweaver-Burke plot has the form y = mx + b,
and is the formula for a straight line

1
V

KM
=
Vmax

[S]

1
Vmax

a plot of 1/V versus 1/[S] will give a straight line with


slope of KM/Vmax and y intercept of 1/Vmax
such a plot is known as a Lineweaver-Burk double
reciprocal plot
Double reciprocal plots can easily be understood and
provide recognizable pattern for the study of ENZYME
INHIBITION

Lineweaver-Burk Plot (Contd)


o KM is the
dissociation
constant for
ES; the greater
the value of
KM, the less
tightly S is
bound to E
o Vmax is the
turnover
number

Turnover Numbers
Vmax is related to the turnover number of
enzyme:also called kcat

V max

turnover _ number kcat


[ET ]
o Number of moles of substrate that react to form
product per mole of enzyme per unit of time

Enzyme Inhibition

Reversible inhibitor: a substance that binds to an enzyme to


inhibit it, but can be released

competitive inhibitor: binds to the active (catalytic) site


and blocks access to it by substrate
noncompetitive inhibitor: binds to a site other than the
active site; inhibits the enzyme by changing its
conformation

Irreversible inhibitor: a substance that causes inhibition that


cannot be reversed

usually involves formation or breaking of covalent


bonds to or on the enzyme

Competitive Inhibition
o Substrate must compete with inhibitor for the
active site; more substrate is required to reach a
given reaction velocity
EI

+ E + S

ES

o We can write a dissociation constant, KI for EI


EI

+ E

KI =

[E][I]
[EI]

Competitive Inhibition

Competitive Inhibition
No inhibition
1 = KM
V
Vmax
y =

1
S

Vmax
b

In the presence of a competitive inhibitor


1 = KM 1 + [I]
V
Vmax
KI

1
S

Vmax
b

In a Lineweaver-Burk double reciprocal plot of 1/V


versus 1/[S], the slope (and the x intercept) changes
but the y intercept does not change

A Lineweaver-Burke Plot for Competitive Inhibition

Noncompetitive Inhibition (Contd)


Several equilibria are involved
+S

E
-I

ES

-S
+I

-I
+S

EI

-S

E + P

+I

ESI

The maximum velocity Vmax has the form

max =

Vmax
1 + [I]/K I

Noncompetitive Inhibition (Contd)

A Lineweaver-Burke Plot for Noncompetitive Inhibition


o Because the inhibitor does not interfere with binding of
substrate to the active site, KM is unchanged
o Increasing substrate concentration cannot overcome
noncompetitive inhibition
No inhibition
1 = KM 1
V
S
Vmax

Vmax

y =
m x + b
In the presence of a noncompetitive inhibitor

1 = KM 1 + [I]
V
Vmax
KI

1
S

Vmax

1 +
b

[I]
KI

A Lineweaver-Burke Plot for Noncompetitive Inhibition


(Contd)

Other Types of Inhibition


Uncompetitive- inhibitor can bind to the ES complex
but not to free E. Vmax decreases and KM decreases.

Mixed- Similar to noncompetitively, but binding of I


affects binding of S and vice versa.

Uncompetitive Inhibition

Uncompetitive Inhibition

Summary
Effects of reversible inhibitors on kinetic constants
Type of Inhibitor

Effect

Competitive (I binds to E
only)

Raises KM; Vmax unchanged

Uncompetitive (I binds to
ES only)

Lowers KM and Vmax

Non-competitive (I binds to
E or to ES)

Lowers Vmax; KM
unchanged

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