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Enzyme Kinetics I

Maud Menten

Leonor Michaelis

Enzyme Kinetics
What is it?
Enzymes are molecular machines as with any machine or mechanistic
process in nature, it is worthwhile to understand:
the individual steps taken by the machine in its process
the rate at which the machine can operate
the environment in which the machine best operates
k1

k2

k-1
Enzyme
(E)

Substrate
(S)

k-2

Enzyme-Substrate
Complex
(ES)

Enzyme
(E)

Product
(P)

Enzyme kinetics is a quantitative approach to understanding the functions of and


mechanisms employed by enzymes.
It is based upon determining the rates at which enzymes catalyze reactions
and the environmental factors that influences these rates.

Why analyze enzyme kinetics?


1. The kinetic analysis of enzyme behavior lends insight into the molecular
mechanisms used by enzymes to convert substrate to product.
2. Kinetic analyses provide both the experimental means and strategy
towards understanding readily measurable quantities that can be used to
describe enzyme behaviour. Quantities derived through enzyme kinetics are
the universal language by which enzymes are compared by biochemists.
3. Enzymes are used in many industrial processes or constitute a component
of useful products. Useful enzymes are identified by their kinetic properties
e.g. their affinity for substrate and the efficiency with which they carry out
reactions.

4. In medicine: many chemicals living organisms may ingest either as


therapeutic drugs or as toxins mediate their effects by binding to enzymes
and altering the rate at which they catalyze reactions.
5. The affinity of an enzyme for substrate and the rate at which it converts
substrate to product is a consequence of its evolved role in the metabolic
pathways that define life.

The kinetic analysis of enzymes preceded knowledge of their molecular


nature
pre 1900
Pasteur posited that the special activities of living cells that could mediate
chemical changes were a special property of life, inseparable from life.
The concept of Vitalism.
around 1900
Eduard Buchner discovered that these chemical activities were the
properties of molecules that could function separate from living
organisms.
The term Enzymes was coined and over the next few decades it was
established that these molecules were proteins.
post 1900
the emergence of theories that described the action of enzymes and their
behavior

reaction rate, v

The rate of an enzyme catalyzed reaction (v) is dependent on substrate


concentration [S]

substrate concentration, [S]


Led Victor Henri in 1903 to propose that reaction proceed via an
interaction between enzyme (E) and substrate (S).

ES complex

in 1913, Leonor Michaelis and Maud Menten proposed a general theory of


enzyme activity:

E+S

fast

slow

k1

k2

k-1

ES

k-2

E+P

reaction rate, v

Vmax

y=

ax
b+x

v=

Vmax[S]
Km + [S]

Vmax is the maximum velocity


of the rate of reaction that occurs
when enzyme is saturated.

KM

substrate concentration, [S]

KM is a measure of the apparent


affinity of the enzyme for substrate

Derivation of the M-M equation

E+S

fast

slow

k1

k2

k-1

ES

k-2

E+P
reaction rates are always measured
as initial velocities, before there is
appreciable depletion of substrate
or formation of product

approximation

E+S

fast

slow

k1

k2

k-1

ES

E+P

-the overall rate of rx (v) is limited by the conversion of ES E + P


-2 factors influence this conversion: rate constant (k2) and concentration of ES, [ES]
therefore, v = k2[ES]

Equation 1

E+S

fast

slow

k1

k2

k-1

ES

E+P

Two important assumptions are made during M-M analysis:


1) substrate is in vast excess i.e. [S] >>[E]
2) steady-state assumption, that ES is forming and breaking down
at same rate.

k1[E][S] = k-1[ES] + k2[ES]


formation

error in text pg 231 line 15 should


read breakdown not formation

breakdown
collect terms on right side equation

k1[E][S] = (k-1 + k2)[ES]


solve for [ES]

[ES] =

k1[E][S]
(k-1 + k2)
next page

E+S

[ES] =

fast

slow

k1

k2

k-1

ES

k1[E][S]
(k-1 + k2)

k1

(k-1 + k2)

1
KM

E+P

since all 3 rate constants are on same side of equation,


we can combine these into one term

or

KM =

(k-1 + k2)

k1

KM is called the Michaelis constant

substitute KM into above equation

[E][S]

[ES] =

KM

Equation 2

next page

E+S

fast

slow

k1

k2

k-1

ES

[E][S]

E+P

[ES] =

KM

Equation 2

the total amount of enzyme, ET, is constant throughout experiment


(i.e. the catalyst is not consumed).
But it may be either free (not bound to substrate), E or bound
to substrate, ES.
therefore,

[ET] = [E] + [ES]

we can solve for [E]

[E] = [ET] - [ES]

and substitute this definition of [E] into Equation 2, so

[ES] =

([ET] [ES])[S]
KM
next page

E+S
[ES] =

[ES] =

fast

slow

k1

k2

k-1

ES

([ET] [ES])[S]
KM

[ET][S] [ES][S]

multiply through by [S]

solve for [ET][S]

KM

[ET][S] = [ES]KM + [ES][S]


[ET][S] = (KM + [S])[ES]

[ES] =

E+P

collect terms

solve for [ES]

[ET][S]
KM + [S]
next page

E+S

[ES] =

fast

slow

k1

k2

k-1

ES

E+P

[ET][S]

remember Equation 1:
then

KM + [S]

v = k2[ES]
v

substitute term for [ES]

v
k2

[ES] =

[ET][S]

k2

solve for v

KM + [S]

k2[ET][S]
KM + [S]

Now, the maximum rate (Vmax) occurs when all enzyme bound to substrate.
That is, when [ET] = [ES]
Under this condition, Equation 1 becomes: Vmax = k2[ET]
substitute in Vmax

v=

Vmax[S]
KM + [S]

the Michaelis-Menten equation

The Significance of Vmax, Km and other kinetic quantities

reaction rate, v

Vmax

y=

ax
b+x

v=

KM

substrate concentration, [S]

Vmax[S]
Km + [S]

Vmax
E+S

fast

slow

k1

k2

k-1

ES

k-2

E+P
reaction rates are always measured
as initial velocities, before there is
appreciable depletion of substrate
or formation of product

approximation

E+S

fast

slow

k1

k2

k-1

ES

E+P

In general the rate of a reaction is dependent on the rate constant k2 and the
substrate concentration. Therefore v = k2[ES]
but, when substrate concentration is so high that all of the enzyme is saturated
then the enzyme is operating as fast as it can and Vmax = k2[ET] where ET is
the total Enzyme in the reaction ([ES]=[ET] when the enzyme is saturated).
Vmax is the maximal velocity of the enzyme

KM
KM is the substrate concentration at which the enzyme is rate is half its maximal
velocity (Vmax) or put another way:
KM is the substrate concentration where the enzyme is half-saturated.

reaction rate, v

Vmax

Vmax

KM

Vmax[S]
v=
Km + [S]

substrate concentration, [S]

On a Michaelis-Menten plot, KM equals the [S] at which the reaction rate


is half-maximal (1/2 Vmax)

KM

k-2

k-1

Vmax[S]

v=
reaction rates are always measured
KM + [S]
as
initial
velocities,
before
there
is
approximation
depletion
of substrate
Km is actually a combined rate constant appreciable
that describes
the formation
and breakdown
or formation of product
of the ES complex
fast

E+S

k1 k 1

slow

k2

ES

(k
k-1-1 + k2)

1
KE
M

+ Por

KM =

(k-1 + k2)

k1

k1
(k-1 + k2)

in most reactions, k2 is usually much lower than k1


therefore Km approximately reduces to:
KM =

k-1
k1

Note that when k1 > k-1, the formation of ES is favored and KM is small
when k-1 > k1, the dissociation if ES is favored and KM is large
that is, small KM indicates high affinity of enzyme for substrate
high KM indicates low affinity of enzyme for substrate

KM is a measure of the affinity of an enzyme for its substrate

1
KM

Kcat
E+S

fast

slow

k1

k2

k-1

ES

E+P

kcat is equivalent to the rate limiting step, e.g. k2


therefore, k2 = kcat

and when all enzyme is bound to substrate,


Vmax = k2[ET]
therefore, k2 =

Vmax
[ET]

= kcat

kcat is also referred to as the turnover number (units of s-1)


that is, the maximum number of substrate molecules per active site
converted to product per second

kcat is the turnover number

kcat
KM

also known as the Specificity constant


the parameters of KM or kcat alone are insufficient for comparing
the overall efficiency of an enzyme
a better measure is to calculate the ratio of kcat to KM
it is a measure of the overall rate of reaction where ultimately every
reaction relies on an encounter of Enzyme with Substrate
the upper limit of kcat/KM is defined by the rate at which E and S
can diffuse together in an aqueous solution
(about 108 to 109 M-1s-1)
some enzymes have specificity constants approaching this value
they are said to be catalytically perfect enzymes
Enzyme

Substrate

Kcat
(s-1)

KM
(M)

kcat/KM
(M-1s-1)

Catalase

H2O2

4 x 107

1 x 100

4 x 107

-lactamase

penicillin

2 x 103

2 x 10-5

1 x108

Practical Determination of Kinetic Parameters in Laboratory

Hypothetical case:
You work for an industrial company that uses the enzyme Cellulase in the
formulation of laundry detergents.
It is your job to determine the kinetic attributes of 5 different Cellulase
enzymes to assess their suitability for use as an additive in laundry detergent.

Background
Cellulase is an enzyme that breaks down Cellulose into glucose molecules

Energy

O2
CO2 + H2O
glucose

cellulose

Cellulases are enzymes that bind


cellulose and chemically cleaves
it back to free glucose molecules
cotton

light

new shirt
surface

wear and tear

after cellulase treatment

Your job
Carry out kinetic analysis of 5 cellulase enzymes
Cellulase 1
Cellulase 2
Cellulase 3
Cellulase 4
Cellulase 5

-determine the most active enzyme of the five.

Measure cellulase enzyme activity by increase in free glucose concentration


The Progress curve of reaction at defined concentration of cellulose, [S].
Enzyme + cellulose Enzyme + glucose

y=mx+b

[glucose]

m=d[glucose]/dt

The rate (v) is the increase


in glucose liberated
from cellulose over time
slope of the line = initial rate
or velocity (v) of the reaction

time

dont confuse this with the


M-M plot, which has axes
of rate vs [S]

Determination of reaction rates at varying substrate concentrations

[S]

multiple progress curves


etc.

[glucose]

[S]=4
[S]=3
[S]=2
[S]=1
time

Next, plot Rates vs [S]

1
2
3
4
5
6

v
0.001
0.002
0.004
0.007
0.010
0.013

Plotting substrate concentration vs rate The Michaelis-Menten curve

v=
rate, v
o
o
o
o

Vmax

o
o
o

Vmax

o
o
o
o
o

[substrate], [S]
KM

Vmax[S]
KM + [S]

So,
Having generated a Michaelis-Menten Plot specific for the Cellulase #1:
You now know the Vmax for the enzyme.
You can now determine KM for the enzyme since KM is the substrate concentration
at 1/2Vmax
Also, you can calculate Kcat since you know how much enzyme you used in the assay
and
Vmax

Kcat =

[ET]

Therefore you can also calculate the specificity constant since

Specificity Constant = Kcat/KM

Enzyme

kcat
(s-1)

KM
(M)

kcat/KM
(M-1s-1)

Cellulase 1

4 x 104

1 x 10-2

4 x 106

Cellulase 2

1 x 102

4 x 10-4

3 x 105

Cellulase 3

2 x 105

4 x 10-2

5 x 106

Cellulase 4

4 x 101

4 x 10-3

1 x 104

Cellulase 5

2 x 103

2 x 10-5

1 x108

Therefore, you would report that Cellulase #5 is the most active enzyme
Note that Cellulase #1 has a higher turnover number (kcat) than Cellulase #5
But Cellulase #5 has a much higher affinity for substrate (KM).
Overall, based on the specificity constant (kcat/KM) Cellulase #5 is the most active
enzyme.

Understand the meanings of


Vmax
the relationship between Vmax and KM
the meaning of Km in terms of substrate affinity

the turnover number, Kcat


Specificity constant Kcat/KM
the general process for determining Vmax and KM in the lab

E+S

fast

slow

k1

k2

k-1

ES

E+P

Recommended Reading
Stryer 8th Edition
Ch 8 Enzymes: Basic Concepts and Kinetics Pgs 216-245.
Stryer 7th Edition
Ch 8 Enzymes: Basic Concepts and Kinetics Pgs 219-248.

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