Michaelis-Menten Kinetics

Enzyme Kinetics
Enzymatic reaction
k1 k2
E+S ES E+P
k-1

Rate expression for product formation

v = dP/dt = k2(ES)
d(ES)/dt = k1(E)(S)-k-1(ES)-k2(ES)

Conservation of enzyme
(E) = (E0) (ES)
 Two Methods to Proceed
Rapid equilibrium assumption: define
equilibrium coefficient
Km = k-1/k1 = [E][S]/[ES]

Quasi-steady state assumption

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

Both methods yield the same final equation

Michaelis- Menten Kinetics
 Michaelis-Menten Kinetics
When v = 1/2 Vmax, [S]= Km so Km is sometimes
called the half-saturation constant and
sometimes the Michaelis constant

Vmax [S ] k 2 [E 0 ][S ]
v
K m [S ] K m [S ]
 Assumptions:
1. The rate of reaction is first order in
concentration of substrate at relatively low
values of concentration
2. As the substrate concentration is continually
increased, the reaction order in substrate
diminishes form one to zero.
3. The rate of reaction is proportional to the total
amount of enzyme present.
 Michaelis-Menten Kinetics

Vmax [S ] k 2 [E 0 ][S ]
v
K m [S ] K m [S ]

units on k2 are amount product per

amount of enzyme per unit time (also
called the turnover number). Units on E0
are amount of enzyme (moles, grams,
units, etc.) per unit volume
Km has the same units as [S] (mole/liter,
etc.)
 Initial velocity Vo
When enzyme is mixed with high concentration of
substrate [S] reaction goes rapidly to steady state.
Does not allow characterization
Use low starting [S] and increase
Hold [enzyme] constant
Measure rate of reaction, Vo as [S] increases
Until rate becomes constant: approaches Vmax
Effect of Substrate
A. Low [S] B. 50% [S] or Km C. High, saturating [S]
Effect of [substrate] on RX Velocity

Vmax = k2 [E]t

[E]t =total enzyme

=[E] +[ES]
Experimentally Determining Rate
Parameters for Michaelis-Menten
Kinetics

Lineweaver-Burk
Eadie-Hofstee
Hanes- Woolf
Batch Kinetics
 Determining Parameters
Rearrange the equation into a linear form.
Plot the data.
What kind of data would we have for an
experiment examining enzyme kinetics?
Describe an experiment.
The intercept and slope are related to the
parameter values.
 Lineweaver-Burk
(double reciprocal plot)
Rewrite Michaelis-Menten rate expression

1 Km 1 1

v Vmax [S ] Vmax
Plot 1/v versus 1/[S]. Slope is Km/Vmax,
intercept is 1/Vmax
Lineweaver-Burk Plot
 Graphical Solution

intercept
1/ V
1 Km 1 1 Slope = Km/ Vmax

v Vmax [S] Vmax
1/ Vmax

-1/ Km 1/ [S]
 Example: Lineweaver-Burk
-5 -5
[S] x 10 M V, M/min x 10
1.0 1.17
1.5 1.50
2.0 1.75
2.5 1.94
3.0 2.10
3.5 2.23
4.0 2.33
4.5 2.42
5.0 2.50
Resulting Plot

slope = Km/ Vmax= 0.5686

y intercept = 1/ Vmax= 2.8687
Michaelis-Menten Kinetics

Vmax [S ] k 2 [E 0 ][S ]
v
K m [S ] K m [S ]
Vmax = 1/2.8687 x 10-4 = 3.49 x 10-5 M/min
Km= 0.5686 x Vm = 1.98 x 10-5 M
 Other Methods
Eadie-Hofstee plot

v
v Vmax K m
[S ]

Hanes- Woolf

[S ] K m 1
[S ]
v Vmax Vmax
Eadie-Hofstee plot
 Hanes-Wilkinson Plot
Another inversion method: multiply MM eq by [S]

v0 = Vmax[S] 1 KM
S/V0 = [S] +
KM + [S] Vmax Vmax

Plot 1/v (= y) versus 1/[S] (= x)

[S]/V
Slope =1/Vmax
-KM
KM/Vmax
[S]
 Example of Michaelis-Menten Enzyme Kinetics
Given this data, what is Vmax? What is KM?
[S] moles/L 1/[S] v (mol/min) 1/v
2.00E-01 5.00 60 0.01666667
2.00E-02 50.00 60 0.01666667
2.00E-03 500.00 60 0.01666667
2.00E-04 5000.00 48 0.02083333
1.50E-04 6666.67 45 0.02222222
1.30E-05 76923.08 12 0.08333333

First, graph [S] vs. v to make sure it obeys MM kinetics

70
v (mol/min)

60

50
Vmax is 60 by inspection
40

30

20

10

0
1.00E-05 1.00E-04 1.00E-03 1.00E-02 1.00E-01 1.00E+00

[S]
 Example of Michaelis-Menten Enzyme Kinetics
Given this data, what is Vmax? What is KM?
[S] moles/L 1/[S] v (mol/min) 1/v
2.00E-01 5.00 60 0.01666667
2.00E-02 50.00 60 0.01666667
2.00E-03 500.00 60 0.01666667
2.00E-04 5000.00 48 0.02083333
1.50E-04 6666.67 45 0.02222222
1.30E-05 76923.08 12 0.08333333

Since Vmax = 60 we can solve for KM, plug this into MM eq.

v0 = Vmax[S] KM = [S] Vmax

-1
KM + [S] v0

If v = 48, [S]= 2 X 10-4, KM = 5.0 X 10-5

If v = 12, [S]= 1.3 X 10-5, KM = 5.2 X 10-5
 Example of Michaelis-Menten Enzyme Kinetics
Given this data, what is Vmax? What is KM?
[S] moles/L 1/[S] v (mol/min) 1/v
2.00E-01 5.00 60 0.01666667
2.00E-02 50.00 60 0.01666667
2.00E-03 500.00 60 0.01666667
2.00E-04 5000.00 48 0.02083333
1.50E-04 6666.67 45 0.02222222
1.30E-05 76923.08 12 0.08333333

We can also check by Lineweaver-Burke plot

0.09

0.08

0.07
1/v 0.06
KM 1 1
0.05 1/Vmax 1/V0 = +
0.04
Vmax [S] Vmax
0.03
-1/KM 0.02
Scale is important
0.01
1/[S]
0
- 40000 - 20000 0 20000 40000 60000 80000 100000
 Comparison of Methods

Lineweaver-Burk: supposedly gives good

estimate for Vmax, error is not symmetric
about data points, low [S] values get more
weight
Eadie-Hofstee: less bias at low [S]
Hanes-Woolf: more accurate for Vmax
 TurnOver Number
kcat is how many reactions an
enzyme can catalyze per
second

Vmax
kcat
ET
For Michaelis -Menton kinetics k2= kcat
When [S] << KM very little ES is formed and [E] = [E]T
and

vo
k2
ET S ES
k cat
KM KM
Kcat/KM is a measure of catalytic efficiency
 What is catalytic perfection?
k1k 2
When k2>>k-1 or the ratio is maximum
k 1 k 2

kcat Or when every substrate that hits

Then
k1 the enzyme causes a reaction to
KM take place. This is catalytic
perfection.
Diffusion-controlled limit- diffusion rate of a substrate
is in the range of 108 to 109 M-1s-1. An enzyme lowers
the transition state so there is no activation energy
and the catalyzed rate is as fast as molecules collide.
What is catalytic perfection?

kcat = Vmax / [E]total

 Batch Kinetics
d[S ] Vmax [S ]
v
dt K m [S ]
integrate
[S 0 ]
Vmaxt [S 0 ] [S ] K m ln
[S ]
rearrange
[S 0 ] [S ] K m [S 0 ]
ln Vmax
t t [S ]
 Inhibited Enzyme Kinetics
Competitive Inhibition
Noncompetitive Inhibition
Uncompetitive Inhibition
Substrate Inhibition
 Experiments: Initial rate at different
substrate concentrations

E S1= 20 E S2=10 E S3=6.7 E S4=5 E S5=4

Measure S for a short time period. Calculate v from:

v = [S(time 0) S(time 1)]/delta time
 Time (min) S (g/L)
Experiment 0 20
Using S1 0.5 19.43

v= (20-19.3)g/L]/0.5 min = 1.14 g/L/min

Time (min) S (g/L)

Experiment 0 10
Using S2 0.5 9.565

v= (10-9.565)g/L]/0.5 min = 0.87 g/L/min

 Experimental Data
S (mmol/L) v (mmol/L/min)
20 1.14
10 0.87
6.7 0.70
5.0 0.59
4.0 0.50

Problems with this method?

Rate is not measured at a constant substrate
concentration substrate decreasing. Must have
sensitive assay for substrate to measure initial rates.
 20
18
16 regression
14 S/v = 0.6S + 5.6
12
S/v (min)

10
8
6 experimental data
regression
4
2
0
0 5 10 15 20 25
S (g/L)
 Allosteric Enzyme Kinetics
In an enzyme with more than one substrate
binding site, binding of one substrate
molecule affects the binding of another.
n
d[S ] V max[S]
v n
dt K m [S ]n

n>1, cooperation; n<1, interference

 Allosteric Inhibition

This term is used to describe the regulation of

enzymes with multiple active sites:

Allosteric inhibition - A molecule binding to the

enzyme causes a conformational change that
decreases the catalytic ability of the enzyme.

Also, allosteric activation is observed in some

enzymes. A molecule binding to the enzyme
causes a conformational change that increases
the catalytic ability of the enzyme.
Allosteric Inhibition
 Allosteric Enzymes
Shape of rate curve is sigmoidal

Michaelis-Menten

Allosteric
 Inhibition of Enzymes
Can be irreversible (metals) or
reversible (product, substrate, salt,
etc.)

1. Competitive
2. Noncompetitive
3. Uncompetitive
 Competitive Inhibition
Inhibitor is an analog of the substrate, and
binds to the active site of the enzyme.
[E ][S ]
K m
E S ES P [ES ]
[E ][I ]
KI
I [EI ]
[E 0 ] [E ] [ES ] [EI ]
EI
v k 2 [ES]
 Competitive inhibition E
ES

(a) A competitive inhibitor (I) binds

to the same site as does the substrate
EI
(S; top).
The inhibitor changes the apparent
Km for the reaction, but not the Vmax
because enough substrate can keep
any inhibitor from binding.
A Lineweaver-Burk plot (bottom)
for the reaction at various
concentrations of the inhibitor
reflects this behavior.
Competitive Inhibition
 Competitive Inhibition
Rate is given by:

Vmax [S ] Vmax [S ]
v
I [S ]
K m,app
K m 1 [S ]

K I

 Competitive Inhibition
1/v I>0

I=0

Vmax is unchanged
1/Vmax

-1/Km -1/Km,app 1/[S]

Competitive Inhibition
 Noncompetitive Inhibition
Inhibitor binds to the enzyme, but not at the
active site. However, the enzyme affinity for
substrate is reduced.

E S ES P [E ][S ] [EI ][S ]

K m
[ES ] [ESI ]
I I
[E ][I ] [ES ][I ]
KI
EI S ESI [EI ] [ESI ]

[E 0 ] [E ] [ES ] [EI ] [ESI ]

v k 2 [ES]
(b) A noncompetitive inhibitor (I) (green) does
not bind to the substrate binding site and can Noncompetitive
bind to both the free enzyme or the ES E ES
complex.
Usually a noncompetitive inhibitor resembles
one substrate (S2) in a two-substrate reaction, EI
as shown here, where both substrates are
present on the enzyme at the same time.
In the simplest cases, noncompetitive
inhibitors don't change the Km for the first
substrate (S), because they don't affect its
binding.
But providing the concentration of S2 is not
high enough to out-compete all the inhibitor,
the inhibitor does reduce the Vmax for the
reaction.
Noncompetitive Inhibition
 Cofactors and Coenzymes
Holoenzymes- three parts
Apoenzyme- Protein portion
Cofactor- inorganic ion (ex: metal ions),
improve the fit of enzyme with substrate
Coenzyme- nonprotein organic molecule
(ex: NAD- nicotinamide adenine
dinucleotide), many synthesized from
vitamins (why vitamins are essential)
 Noncompetitive Inhibition
Rate is given by:

Vmax Vmax,app
v
[I ] K m K m
1 1 1

K I


[S ]

[S ]

 Noncompetitive Inhibition
1/v I>0

I=0

1/Vmax,app

1/Vmax

-1/Km 1/[S]
Km is unchanged
 Uncompetitive Inhibition
Inhibitor binds only to ES complex, and not
to E alone.
E S ES P [E ][S ]
K m
[ES ]
I
[E ][I ]
KI
[EI ]
ESI
[E 0 ] [E ] [ES ] [ESI ]

v k 2 [ES]
 Uncompetitive inhibition

(c) An uncompetitive inhibitor

(blue) binds only to the enzyme-
substrate (ES) complex and slows
down the reaction probably by
inducing a conformational change
in the enzyme.
Both the apparent Km and Vmax are
affected proportionally by such an
inhibitor, leading to parallel
Lineweaver-Burk plots for
different inhibitor concentrations.
 Uncompetitive Inhibition
Rate is given by:

Vmax
[S ]
[I ]
1
K I Vmax,app [S ]
v
K m K [S ]
[S ] m ,app
[I ]
1

K I
 Uncompetitive Inhibition
1/v
I>0

I=0
1/Vmax,app

1/Vmax

-1/Km,app -1/Km 1/[S]

 Substrate Inhibition
No substrate inhibition
v

Substrate inhibition
Vmax [S ]
v
[S]2
K m [S ]
KSi

[S ]max. rate K mK Si S
Figure U2-4.1 Competitive, noncompetitive and
uncompetitive inhibition
 Enzyme reactions can be slowed by the presence of
inhibitors
A key parameter that can be obtained from such an analysis is the
affinity of the inhibitor for the enzyme, the inhibition constant Ki.
By convention, Ki is given as the dissociation constant for the enzyme-
inhibitor equilibrium:

Ki = [E][I] Ki = [ES][I]
[EI] [ESI]
The lower the value of Ki the tighter the inhibitor binds. In
pharmacology, the value of Ki is often used as a measure of the
effectiveness of a drug.
A compound with a very low Ki, say 10-9 M (nanomolar) or less, can
be given at very low doses and will still be able to bind its target.
Enzyme Inhibitor Classification
1. Competitive inhibitors:
Most common class of reversible inhibitor consists of
compounds that resemble the substrate.
Such molecules can fit into the substrate binding site,
thereby blocking access from substrate molecules.
These inhibitors compete with the substrate for the
active site.
Example: Many HIV protease inhibitors that have
proven to be effective in treatment of AIDS are
competitive inhibitors that were designed to resemble
the peptide substrate of the HIV protease.
Enzyme Inhibitor Classification
Not all inhibitors compete with the substrate for the
active site of the enzyme; other inhibitors bind to a
separate site.

2. Noncompetitive inhibitors bind to both the free

enzyme and the enzyme-substrate complex.
If an enzyme has two substrates that must bind
simultaneously for the reaction to occur, an inhibitor
might compete with one substrate but not the other.

Small Km means tight binding; high Km means

weak binding
 Enzyme Inhibitor Classification
3. Uncompetitive inhibitors bind only to the enzyme-
substrate complex.
In effect, the inhibitor reduces the amount of ES that
can go on to form product.
Uncompetitive inhibitors often stabilize an alternative
conformation of the protein, and in this case they are
called allosteric inhibitors.
There are also allosteric activators: molecules that
activate enzymes by stabilizing a conformation of the
enzyme that is more active than the conformation that
exists in their absence.
Dihydrofolate reductase (DHFR) is an enzyme that
reduces 7,8-dihydrofolate (DHF) to 5,6,7,8
tetrahydrofolate , a step in the biosynthesis of
thymidine. Methotrexalate (MTX), a structural
analogue of dihydrofolate, inhibits this enzyme. MTX
has been used in cancer therapies because inhibition of
DHFR restricts the thymidine production required for
cell division. In the absence of thymidine, the
cancerous cells cannot multiply. Using the following
data of DHFR activity in the presence of MTX,
determine the inhibition mechanism of MTX and the
maximum reaction rate. Finally, if Km for DHFR is
0.100 M, what is Ki?
 DHF, mM Rate with 50 Rate with 100 Rate with 200
nM MTX, nM MTX, nM MTX,
mM/s mM/s mM/s
3 7.38 5.56 3.72
6 8.84 7.38 5.55
9 9.46 8.29 6.65
12 9.79 8.84 7.38

Using LINEWEAVER-BURK Equation

Plot 1/rate (y) vs 1/[DHF] (x)

 0.3

y = 0.5331x + 0.0912
0.25
y = 0.2668x + 0.091
R = 1
0.2 50nM
100nM
200nM
0.15
Linear (50nM)
Linear (100nM)
y = 0.1337x + 0.0909
0.1 Linear (100nM)
Linear (200nM)

0.05

0
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35
The variation in slope and common y-intercept for the
reciprocal plot as a function of MTX concentration
demonstrates that MTX is a COMPETITIVE
INHIBITOR. Best fit by a straight line to the 200 nM
data corresponds to a y-intercept of 0.091mM/s and
slope of 0.535 s.

Vmax = 10.9890 mM/s K*M = 5.8791 mM

KI = 3.40 pM

The small value of KI demonstrates that MTX binds

strongly to DHR
 Enzyme Deactivation
Enzymes are denatured by
Temperature
pH
Radiation
Irreversible binding by inhibitors
Temperature can both increase
(thermal activation) and decrease
(thermal denaturation) rate
 Temperature effects
At moderate temperatures, higher
temperatures give higher rates.
E a
v k 2 [E ], where k 2 Ae RT

At higher temperatures, rate starts to

decrease as enzyme denatures faster
d[E ] E d
k d t
k d [E ], or [E] [E 0 ]e , where kd Ad e d RT
dt
 Temperature Effects
Effect on rate is a combination of the two effects

E a
k d t
v Ae RT
[E 0 ]e

Activation energy 10
kcal/mol
Deactivation energy 100
kcal/mol