Enzyme Kinetics

15-Feb-16
Biochemical Engineering
CHE F421
BITS Pilani
Pilani Campus
Amit Jain, Ph.D.

Dept. of Chemical Engg.
BITS Pilani Pilani Campus
BITS Pilani
Pilani Campus
Enzyme Kinetics
15-Feb-16
Enzymes : Introduction
3
Globular proteins (MW >15000 Daltons)

Catalytically active biological macromolecules
Specific, Versatile, Efficient, & are active in aqueous
solution.
Some RNA also catalyze reactions & are called

ribozymes.
Enzymes are named by adding suffix -ase to
the end of:
Substrate. Eg. Urease.
Reaction catalyzed. Eg. Alcohol hydrogenase,
oxidoreductases, etc.
amitjain@pilani.bits-pilani.ac.in
Enzymes : Introduction
4
Some protein enzymes (holoenzymes) require a

non protein group for their activity:
Cofactor, such as metal ions Mg, Zn, Mn, Fe;
Coenzyme, such as complex organic molecule
NAD, FAD, CoA, etc.
Some Vitamins. Eg. Riboflavin.
Holoenzyme = apoenzyme (protein part) +

Cofactor
Isozymes: enzymes with several molecular
forms, but catalyze the same reaction.
15-Feb-16
Enzyme Catalysis
5
Enzymes DO NOT change the equilibrium

constant of a reaction.
Enzymes DO NOT alter the amount of energy
consumed or liberated in the reaction (standard
free energy change, G)
Enzymes DO increase the rate of reactions that
are otherwise possible.
Enzymes DO decrease the activation energy of
a reaction ( GA)
Activation Energy
6
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Example: Hydrogen peroxide

7
Uncatalyzed Rxn. at 20oC:

Ea=18 kcal/mol
Chemically catalyzed (colloidal platinum):

Ea=13 kcal/mol
Enzymatically catalyzed:
Ea=7 kcal/mol
Catalase accelerates the rate of rxn. by a factor

of 108 (Prove it ???)
The ratio of rates is: exp(-7000/2x293)/exp(-18000/2x293)
Enzyme Catalysis: ES Complex

8
ES complex revealed x-ray and Raman

spectroscopy.
ES interaction weak forces:
Van der Waals, H-bonding, Ionic bonding
The substrate is a relatively small molecule and

fits into a certain region on the enzyme.
Simplest model describing E-S interaction is the
lock and key model.
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Spectroscopic Analysis
9
(d) Reaction scheme showing 2-HMS oxidation by AMSDH.

(e) Representative assay showing the activity of AMSDH (200nM) on
2-HMS (max 375nm) in 50s. The inset is a MichaelisMenten plot.
[Source: Nature Communications 6, Article number: 5935 doi:10.1038/ncomms6935]
ES Complex: Lock & Key Model

10
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Factors Affecting Enzyme

Catalysis
11
Multisubstrate enzyme-catalyzed reactions

Proximity effect
Substrates reactive regions and enzymes active site are
brought closer
Orientation effect
Improved reaction rate at certain positions and angles
Induced fit
Formation of ES complex causes slight change in 3-D shape
of the enzyme
Enzyme structure (Primary/sec./ter./quat.)

Properties of active site
Folding characteristics
Enzyme Kinetics
12
Study the rate of enzyme catalyzed reactions.

Models for enzyme kinetics
Michaelis-Menten kinetics
Inhibition kinetics
Effect of pH and Temperature
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Enzyme Kinetics
13
Mathematical model of
single substrate enzyme
catalyzed reactions:
V.C.R Henri 1902
Michaelis and Menten
1913
M-M Kinetics or saturation
kinetics
Similar to LangmuirHinselwood kinetics
Saturation Enzyme Kinetics

This model is based on data from batch reactors
with constant liquid volume.
Initial substrate, [S0] and enzyme [E0]
concentrations are known.
An enzyme solution has a fixed number of
active sites to which substrate can bind.
At high substrate concentrations, all these sites
may be occupied by substrates or the enzyme is
saturated.
BITS Pilani, Pilani Campus
15-Feb-16
Initial Velocity (vo) and [S]
15
The concentration of substrate [S] present will

greatly influence the rate of product formation,
termed the velocity (v) of a reaction.
Studying the effects of [S] on the velocity of a
reaction is complicated by the reversibility of
enzyme reactions, e.g. conversion of product back
to substrate.
To overcome this problem, the use of initial velocity
(vo) measurements are used. At the start of a
reaction, [S] is in large excess of [P], thus the initial
velocity of the reaction will be dependent on
substrate concentration.
Initial Velocity (vo) and [S]

(cont)
16
When initial velocity is

plotted against [S], a
hyperbolic curve results,
where Vmax represents
the maximum reaction
velocity.
At this point in the
reaction, if [S] >> E, all
available enzyme is
"saturated" with bound
substrate, meaning only
the ES complex is
present.
15-Feb-16
Substrate Saturation of an
Enzyme
A. Low [S]
17
B. 50% [S] or Km C. High, saturating [S]

M-M Enzyme Kinetics

18
Saturation kinetics can be obtained from a simple

reaction scheme that involves a reversible step for
enzyme-substrate complex formation and a
dissociation step of the ES complex.
K1
E+S
K-1
k2
ES
P + E
where the rate of product formation v (moles/l-s,

d[P ]
g/l-min) is
v =
= k 2 [ ES ]
dt
Ki is the respective reaction rate constant.
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M-M Enzyme Kinetics

19
The rate of variation of ES complex is
d[ES]
= k1[E][S] k1[ES] k2[ES]
dt
Since the enzyme is not consumed, the
conservation equation on the enzyme yields
[ E ] = [ E0 ] [ ES ]
M-M Enzyme Kinetics

20
How to use independent variable [S] to represent v ?
v=
d [ P]
= k 2 [ ES ]
dt
[ E ] = [ E0 ] [ ES ]
d[ ES ]
= k1[ E][S ] k 1[ ES ] k2[ ES ]
dt
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Approaches
21
At this point, an assumption is required to

achieve an analytical solution.
The rapid equilibrium assumption
Michaelis - Menten Approach.
The quasi-steady-state assumption.

Briggs and Haldane Approach.
Michaelis - Menten Approach

22
The rapid equilibrium assumption:

Assumes a rapid equilibrium between the enzyme
and substrate to form an [ES] complex.
K1
E+S
K-1
k2
ES
P + E
k1[ E ][ S ] = k 1[ ES ]
' can be expressed by the
The equilibrium constant K m
following equation in a dilute system.
' = k 1 = [ E ][S ]
Km
k1
[ ES ]
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15-Feb-16

23
Then rearrange the above equation,
[ ES ] =
[ E ][ S ]
K m'
Substituting [E] in the above equation with enzyme

mass conservation equation
[ E ] = [ E 0 ] [ ES ]
yields,
[ ES ] =
([ E0 ] [ ES ])[ S ]
K m'

24
[ES] can be expressed in terms of [S],
[ ES ] =
[ E0 ][ S ]
K m' + [ S ]
Then the rate of production formation v can be

expressed in terms of [S],
k [ E 0 ][ S ]
Vm [ S ]
d[P]
v=
= k [ ES ] = 2
=
2
' + [S ]
' + [S ]
dt
Km
Km
Where, Vm = k [ E0 ] represents the maximum forward rate
2
of reaction (e.g. moles/L-min).
12
15-Feb-16

25
K m' is often called the Michaelis-Menten constant,

mol/L, mg/L.
The prime reminds us that it was derived by assuming
rapid equilibrium in the step of enzyme-substrate
complex formation.
k
A low value of Km (K m' = 1 ) indicates affinity of
k
enzyme to the substrate. 1
It corresponds to the substrate concentration, giving
the half-maximal reaction velocity, i.e.,
v=
Vm [S ]
Vm [S ]
1
=
= Vm
'
K m + [S ] [S ] + [S ] 2
Briggs-Haldane Approach
26
The quasi-steady-state assumption:

- A system (batch reactor) is used in which the
initial substrate concentration [S0] greatly
exceeds the initial enzyme concentration [E0].
since [E0] was small,
d[ES]/dt 0
- It is shown that in a closed system the quasisteady-state hypothesis is valid after a brief
transient if [S0]>> [E0].
13
15-Feb-16
Quasi-steady-state
Assumption
27
The quasi-steadystate hypothesis is

valid after a brief
transient in a batch
system if [S0]>>
[E0].
28
With such assumption, the equation representing the

accumulation of [ES] becomes
d [ ES ]
= k1[ E ][S ] k1[ ES ] k2[ ES ] 0
dt
Solving this algebraic equation yields
[ ES ] =
k1[ E ][ S ]
k 1 + k 2
14
15-Feb-16
29
Substituting the enzyme mass conservation equation
[ E ] = [ E 0 ] [ ES ]
in the above equation yields
k ([ E ] [ ES ])[ S ]
[ ES ] == 1 0
k 1 + k 2
Using [S] to represent [ES] yields
[ ES ] ==
[ E 0 ][ S ]
k 1 + k 2
+ [S ]
k1
30
Then the product formation rate becomes
v=
k 2 [ E0 ][S ]
d [ P]
= k 2 [ ES ] =
k 1 + k 2
dt
+ [S ]
k1
Then,
v=
Vm [ S ]
K m + [S ]
Where Vm = k [ E0 ] same as that for rapid equilibrium assumption.

2
k + k 2 when K2 << k-1, K m = K m ' = k 1
K m = 1
k1
k
15
15-Feb-16
31
Michaelis-Menten
Briggs-Haldane
d[ES]/dt 0
Assumption: k [E][S] = k [ES]

1
1
Equation:
v=
Vm [ S ]
' + [S ]
Km
Maximum forward
reaction rate:
Vm = k 2 [ E0 ]
Constant:
' = k 1
Km
k1
when k2 << k-1,
v=
Vm [ S ]
K m + [S ]
Vm = k 2 [ E0 ]
k + k2
K m = 1
k1
k + k2
K m = K m ' = 1
k1
Experimental Determination of
Rate Parameters for M-M Kinetics
32
Experimentally determining rate parameters for

Michaelis-Menten type kinetics,
v=
Vm [ S ]
K m + [S ]
To determine the rate parameters:

Predict a specific enzyme catalysis system.
Design bioreactor
16
15-Feb-16
33
The determination of Vm and Km are typically

obtained from initial-rate experiments.
A batch reactor is charged with known initial concentration
of substrate [So] and enzyme [Eo] at specific conditions
such as T, pH, and Ionic Strength.
The product or substrate concentration is plotted against
time.
The initial slope of this curve is estimated.
v=(d[P]/dt) , or = - (d[S]/dt) .
This value v depends on the values of [E0] and [S0].
Many such experiments can be used to generate many
pairs of V and [S] data, these data can be plotted as v-[S].
Initial-rate Experiments
34
17
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35
Accurate determination of Km from initial rate

plots is very difficult.
Other suggested methods for determination of
Vm and Km are:
Double-reciprocal plot (Lineweaver-Burk plot)

Eadie-Hofstee plot
Hanes-Woolf plot
Batch kinetics
Double-reciprocal plot
(Lineweaver-Burk plot)
v=
36
Vm [ S ]
K m + [S ]
Linearizing it in
double-reciprocal form:
K 1
1
1
=
+ m
v Vm
Vm S
18
15-Feb-16
Double-reciprocal plot
(Lineweaver-Burk plot)
37
Slope equals to Km/Vm

y-intercept is 1/Vm.
More often used as it shows the independent
variable [S] and dependent variable v.
1/v approaches infinity as [S] decreases
Gives undue weight to inaccurate measurement
made at low concentration
Give insufficient weight to more accurate
measurements at high concentration.
Eadie-Hofstee Plot
38
v = Vm K m
v
[S ]
the slope is Km
y-axis intercept is Vm.
Can be subject to large error
since both coordinates contain
dependent variable v,
But there is less bias on points at
low [s].
19
15-Feb-16
Hanes-Woolf (Langmuir) Plot

39
[S ] K m
1
=
+
[S ]
v
Vm Vm
the slope is 1/Vm
y-axis intercept is
Km/Vm
Better fit: even
weighting of the data
Used to determine Vm
more accurately
Batch Kinetics
40
The time course of variation of [S] in a batch

enzymatic reaction can be determined from:
v=
V [S ]
d [S ]
= m
dt
K m + [S ]
by integration to yield
Vm t = [S 0 ] [S ] + K m ln
[S0 ]
[S ]
20
15-Feb-16
Batch Kinetics
41
or
Vm
[S0 ] [S ] = K m ln [S0 ]
[S ]
t
t
A plot of 1/t ln[S0]/[S] versus {[S0]-[S]}/t results in a

line of slope -1/Km and intercept of Vm/Km
Interpretation of Km and Vm
42
Vm
The unit of Vm is the same as that of a reaction
rate: (moles/l-min, g/l-s)
The dimension of K2 must reflect the units of [E0]
Vm = k [ E0 ]
2
If the enzyme is highly purified, it may be

possible to express [E0] in mol/l, g/l, then K2 in
1/time.
21
15-Feb-16
43
If the enzyme is crude, its concentration is expressed

in units.
A unit is the amount of enzyme that gives a
predetermined amount of catalytic activity under
specific conditions.
One unit would be formation of one mol product per
minute at a specific pH and temperature with a
substrate concentration much greater than the value
of Km.
If Vm is mmol/ml-min, [E0] is units/ml, then K2 should
be in mmol/unit-min.
44
Specific Activity is the number of units of activity

per amount of total protein.
Ex. A crude cell lysate might have a specific
activity of 0.2 units/mg or ml protein which upon
purification may increase to 10 units/mg or ml
protein.
pH extremes or temperature during purification
may result in enzyme denaturation (loss of
catalytic activity).
22
15-Feb-16
45
To be Continued
46
Next Class:
Models for more complex enzyme kinetics
Allosteric enzymes
Inhibited enzyme kinetics
Effects of pH and Temperature
Insoluble Substrates
23
15-Feb-16
47
Thanks for participation !!
24

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15-Feb-16

Amit Jain, Ph.D.

Globular proteins (MW >15000 Daltons)

Some RNA also catalyze reactions & are called

Some protein enzymes (holoenzymes) require a

Holoenzyme = apoenzyme (protein part) +

Enzymes DO NOT change the equilibrium

Example: Hydrogen peroxide

Uncatalyzed Rxn. at 20oC:

Chemically catalyzed (colloidal platinum):

Catalase accelerates the rate of rxn. by a factor

The ratio of rates is: exp(-7000/2x293)/exp(-18000/2x293)

Enzyme Catalysis: ES Complex

ES complex revealed x-ray and Raman

The substrate is a relatively small molecule and

(d) Reaction scheme showing 2-HMS oxidation by AMSDH.

ES Complex: Lock & Key Model

Factors Affecting Enzyme

Multisubstrate enzyme-catalyzed reactions

Enzyme structure (Primary/sec./ter./quat.)

Study the rate of enzyme catalyzed reactions.

Effect of pH and Temperature

Saturation Enzyme Kinetics

BITS Pilani, Pilani Campus

Initial Velocity (vo) and [S]

The concentration of substrate [S] present will

Initial Velocity (vo) and [S]

When initial velocity is

B. 50% [S] or Km C. High, saturating [S]

M-M Enzyme Kinetics

Saturation kinetics can be obtained from a simple

where the rate of product formation v (moles/l-s,

M-M Enzyme Kinetics

The rate of variation of ES complex is

M-M Enzyme Kinetics

How to use independent variable [S] to represent v ?

At this point, an assumption is required to

The quasi-steady-state assumption.

Michaelis - Menten Approach

The rapid equilibrium assumption:

Michaelis - Menten Approach

Then rearrange the above equation,

Substituting [E] in the above equation with enzyme

Michaelis - Menten Approach

[ES] can be expressed in terms of [S],

Then the rate of production formation v can be

Michaelis - Menten Approach

K m' is often called the Michaelis-Menten constant,

The quasi-steady-state assumption:

The quasi-steadystate hypothesis is

With such assumption, the equation representing the

Substituting the enzyme mass conservation equation

Then the product formation rate becomes

Where Vm = k [ E0 ] same as that for rapid equilibrium assumption.

Assumption: k [E][S] = k [ES]

Experimentally determining rate parameters for

To determine the rate parameters:

The determination of Vm and Km are typically

Accurate determination of Km from initial rate

Double-reciprocal plot (Lineweaver-Burk plot)

Slope equals to Km/Vm

Hanes-Woolf (Langmuir) Plot

The time course of variation of [S] in a batch

A plot of 1/t ln[S0]/[S] versus {[S0]-[S]}/t results in a