Enzyme Kinetics-2

Dr.Saba Abdi

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 Enzyme Kinetics
Enzyme Kinetics:
Study the rate of enzyme catalyzed
reactions to understand how enzyme
functions.

- Models for enzyme kinetics

- Michaelis-Menten kinetics
- Inhibition kinetics

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Saturation
Enzyme
Kinetics
At high [S], the
enzyme is said to
be saturated with
respect to
substrate

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 Michaelis-Menten kinetics or
saturation kinetics
L. Michaelis and M.L. Menten in 1913 proposed a simple
model to account for kinetic characteristics of enzymes
The critical feature is that a specific ES complex is a
necessary intermediate in catalysis.

Consider an enzyme that catalyzes the S to P by the

following pathway:
k1 k2

E + S ES E+P
k-1 k-2

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Initial Velocity (vo )- determined from slope of
the curve at beginning of the reaction

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Initial velocity is
measured at begnning
of reaction when very
little P has been
made.
So, the reverse
reaction is
insignificant.

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 M-M Enzyme Kinetics
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
k2
E+S
K-1
ES P + E
where the rate of product formation v (moles/l-s, g/l-min) is
d[P]
v = = k 2
[ ES ]
dt
Ki is the respective reaction rate constant.
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 Enzyme Kinetics
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 ]
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 Enzyme Kinetics

d [ P]
v= = k [ ES ]
dt 2

[ E ] = [ E0 ] [ ES ]

d[ ES ]
= k1[ E ][S ] k 1[ ES ] k 2[ ES ]
dt

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

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 Enzyme Kinetics
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.

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 Michaelis - Menten Approach

The rapid equilibrium assumption:

- Assumes a rapid equilibrium between the
enzyme and substrate to form an [ES]
complex.
K1
k2
E+S K-1
ES P + E

k1[ E ][S ] = k 1[ ES ]

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 Michaelis - Menten Approach
The equilibrium constant K m' can be expressed by
the following equation in a dilute system.

K1
k2
E+S ES P + E
K-1

' k 1 [ E ][ S ]
Km = =
k1 [ ES ]

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 Michaelis - Menten Approach
Then rearrange the above equation,
[ E ][ S ]
[ ES ] ==
Km '

Substituting [E] in the above equation with enzyme

mass conservation equation
[ E ] = [ E 0 ] [ ES ]

yields,
([ E0 ] [ ES ])[ S ]
[ ES ] ==
Km '
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 Michaelis - Menten Approach
[ES] can be expressed in terms of [S],
[ E0 ][ S ]
[ ES ] ==
Km' + [S ]

Then the rate of production formation v can be

expressed in terms of [S],
d[P] k 2 [ E 0 ][ S ] Vm [ S ]
v= = k 2 [ ES ] = =
dt Km ' + [S ] Km' + [S ]
Where Vm = k 2 [ E0 ]
represents the maximum forward rate of reaction (e.g.moles/L-min
14 ).
 Michaelis - Menten Approach
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 1
K m =
k1

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 Briggs-Haldane Approach
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 quasi-
steady-state hypothesis is valid after a brief
transient if [S0]>> [E0].
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The quasi-steady-state hypothesis is valid after a
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brief transient in a batch system if [S0]>> [E0].
 STEADY STATE
The more ES present, the faster ES
will dissociate into E + P or E + S.
Therefore, when the reaction is started by
mixing enzymes and substrates, the [ES]
builds up at first, but quickly reaches a
STEADY STATE, in which [ES] remains
constant. This steady state will persist until
almost all of the substrate has been
consumed.
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 Briggs-Haldane Approach
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

k1[ E ][ S ]
[ ES ] ==
k1 + k2
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 Briggs-Haldane Approach
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

[ E 0 ][ S ]
[ ES ] ==
k 1 + k 2
+ [S ]
k1
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 Briggs-Haldane Approach
Then the product formation rate becomes
d [ P] k 2 [ E0 ][S ]
v= = k [ ES ] =
dt 2 k 1 + k 2
+ [S ]
k1

Then, Vm [ S ]
v=
K m + [S ]
Where Vm = k [ E0 ] same as that for rapid equilibrium assumption.
2

k 1 + k 2 when K2 << k-1, K m = K m ' = k 1

Km = k1 21
k 1
 Comparison of the Two
Approaches
Michaelis-Menten Briggs-Haldane
Assumption: k1[E][S] = k1[ES] d[ES]/dt 0

Vm [S ] Vm [ S ]
Equation: v= v=
Km' + [S ] K m + [S
[S ]
Maximum forward
Vm = k 2 [ E0 ] Vm = k [ E0 ]
reaction rate: 2

Constant:
k 1 + k 2
' k 1 Km =
Km = k1
k1
k 1 + k 2
when k2 << k-1, Km = Km '=
k1 22
 Km
Km is the [S] at 1/2 Vmax Km is a constant for a
given enzyme
Km is an estimate of the equilibrium constant for
S binding to E
Small Km means tight binding; high Km means
weak binding Km is the [S] at 1/2 Vmax
Km is a constant for a given enzyme
Km is an estimate of the equilibrium constant for
S binding to E
Small Km means tight binding; high Km means
weak binding
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 Understanding Vmax-
Vm =k2[E0]
The theoretical maximal velocity Vmax is a constant for a
given enzyme. Vmax is the theoretical maximal rate of
the reaction - but it is NEVER achieved .To reach Vmax
would require that ALL enzyme molecules have tightly
bound substrate
- 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]
-if the enzyme is highly purified, it may be possible to
express [E0] in mol/l, g/l, then K2 in 1/time.

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 The turnover number
The kcat is a direct measure of the
catalytic production of product under
saturating substrate conditions.
kcat, the turnover number, is the
maximum number of substrate molecules
converted to product per enzyme molecule
per unit of time.
According to M-M model, kcat = Vmax/Et
Values of kcat range from less than 1/sec
to many millions per sec 25
 The catalytic efficiency

It shows what the enzyme can accomplish

when abundant enzyme sites are
available.
It is the kcat/Km value that allows direct
comparison of the effectiveness of an
enzyme toward different substrates.

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Measuring Km and Vmax
Lineweaver-Burk Plot (Double-Reciprocal Plot)

Vm [ S ]
v=
K m + [S ]

Linearizing it in double-reciprocal form:

1 1 Km 1
= +
v Vm Vm S

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- Lineweaver-Burk plot (Double-reciprocal plot)

- a 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

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 Eadie-Hofstee Plot
v
v = Vm K m
[S ]

- the slope is Km
- y-axis intercept is Vm.
-Can be subject to large error since both coordinates contain
dependent variable v,
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but there is less bias on points at low [s].
 Hanes-Woolf (Langmuir) Plot

[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 32
 Enzyme Inhibition
Inhibitor: Any molecule that acts directly
on an enzyme to lower its catalytic rate.
These can be cellular metabolites, or
foreign substances such as drugs or toxins
that have either a therapeutic or toxic (can
be lethal) effect.
Reversible: Competitive with the
substrate ,Non-competitive with the
substrate, uncompetitive and mixed
inhibitor
Irreversible (I is covalently bound, in 33
capacitating the enzyme)
 Irreversible Inhibition: inhibitor binds tightly, often
covalently, to the enzyme, permanently inactivating it

DIPF = DIFP =
diisopropylfluorophosphate
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Reversible Inhibitors

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 Reversible Inhibition: Competitive
Inhibitor resembles substrate but cant
undergo the catalytic step, so it wastes the
enzymes time by preventing S binding.
i.e. Inhibitor COMPETES with substrate for
binding.
k1 Kcat
E+S ES E+P

+ k-1

I
Ki
EI
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 Competitive inhibitor
In the presence of a competitive inhibitor, I,
Vmax [S]
v0 =
Km(1 + [I]/Ki) + [S]

[E][I]

where Ki (inhibition constant) =

[EI]

Then, Vmax [S]

v0 =
Km+ [S]

where = (1 + [I]/Ki)

The type of inhibition can be determined using the

double reciprocal plot. 37
In competitive inhibition, inhibition can be overcome by high [S].

Vmax does not change, but Km increases (Km,app = Km).

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Competitive inhibitor

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Methotrexate- A competitive inhibitor of
dihydrofolate reductase - role in purine
& pyrimidine biosynthesis, Is used to treat cancer

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Tetrahydrofolate-Substrate for
dihydrofolate reductase

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 Competitive Inhibitor

COO OOC H
CH2 succinate dehydrogenase
CH2
H COO
COO
Fumarate
Succinate

COO
CH2 succinate dehydrogenase
No reaction
COO

Malonate

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 Reversible Inhibition: Non-
Competitive
A molecule or ion binds at a remote site on
the enzyme in such a way that it affects
kcat.
k1 Kcat

E+S ES E+P
+ k-1 +
I I
Ki k1 Ki

EI + S ESI
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k-1
44
 noncompetitive inhibitor
Ex) Compulsory ordered Bi-Bi reaction.

B -BX
E + AX
X EAX EAXB EABX EA E + A

B
EAXI EAXIB

Compound, AXI is a noncompetitive inhibitor of B.

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 Uncompetitive Inhibitor
An uncompetitive inhibitor binds at a site
other than the active site The inhibitor
does not bind free E, but binds E-S
complex and forms an inactive E-S-I
complex which cannot give normal
product.
ES + I ESI

Inhibitor affects both Vmax and Km.

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Uncompetitive Inhibitor

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 Uncompetitive Inhibitor
v0 = Vmax [S]
Km + [S]

where = (1 + [I]/Ki)

and Ki = [ES][I]/[ESI].

Since I does not share the

binding site with S,
uncompetitive inhibition cannot
be overcome by high [S].

Vmax,app decrease
(by a factor of -1)
Km,app decrease
(by a factor of -1)
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 Rare in single-substrate reaction.
More common in multisubstrate reaction

Ex) Compulsory ordered Bi-Bi reaction.

B BX

E + AX
X EAX EAXB EABX EA E + A

EAXBI No reaction

Compound, BI is an uncompetitive inhibitor of AX.

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Mixes Inhibition
Inhibitor binds at a
site other than the
active site (E or ES)
and causes changes
in the overall 3-D
shape of the enzyme
that leads to a
decrease in activity:

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 Mixed inhibition cannot be overcome by high [S].
Vmax,app decrease (by a factor of (1 + [I]/Ki))
Km,app unchanged

Vmax[S]
v0 =
Km + [S]

where = (1 + [I]/Ki) and

= (1 + [I]/Ki)
Ki = [E][I]/[EI],
Ki = [ES][I]/[ESI].
When, = , that is, I binds to E
and ES with the same affinity
(Ki = Ki) Noncompetitive
inhibition.
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 Enzyme Inhibition
Inhibitor Type Binding Site on Enzyme Kinetic effect
Competitive Specifically at the catalytic site, where it Vmax is unchanged;
Inhibitor competes with substrate for binding in a Km, is increased
dynamic equilibrium- like process.
Inhibition is reversible by substrate

Noncompetitive Binds E or ES complex other than at the Km appears unaltered;

Inhibitor catalytic site. Substrate binding Vmax is decreased
unaltered, proportionately to
but ESI complex cannot form products.
inhibitor concentration
Inhibition cannot be reversed by
substrate
Uncompetitive Binds only to ES complexes at Apparent Vmax
Inhibitor locations other than the catalytic site. decreased;
Substrate binding modifies enzyme Km, is decreased
structure, making inhibitor- binding site
available. Inhibition cannot be reversed
by substrate

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.

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 USES OF INHIBITORS
As drugs (in pharmacology)
Agricultural pesticides and insectidies.
In study of Mechanism of E action.
In diagnosis of disease e.g.
e.g. In prostrate cancer in men Acid phosphatase
L-tartrate inhibits competitively 95% of the acid phosphate from
prostrate, but has lower inhibitory effect on acid
phosphatasefrom other sources. Samples from suspected
carcinoma patients can be assessed in presence and absence
of L-tartrate.
Examples of I as drugs:
Antiviral drugs
Antibacterial drugs

Anti tumor drugs e.g. sulfa drugs Antibacterial drugs

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