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Fundamentals of Enzyme Kinetics

Department of Biochemistry
History of Enzymes
-Enzymes are biological catalysts.
-Each enzyme has a genetically
determined and unique primary
sequences.

History of enzyme researches
-In 1700s, studies of meat digestion by stomach secretions
-In 1800s, studies of conversion of starch into sugar by saliva
and plant extract
-In 1850s, Louis Pasteur concluded that the fermentation of
sugar into alcohol is catalyzed by ‘ferments’.
-In 1897s, Buchner discovered that yeast extract could ferment
sugar into alcohol and proved that the fermentation can be
promoted by molecules that continued to function when it was
separated from the cells.
History of Enzymes

History of enzyme researches
-In 1897s, Kuhne called these molecules that can catalyst the
biological reaction as enzymes.
-In 1926, Sumner isolated and crystallized urease and found
that enzymes are proteins.
-Northrop and Kunitz isolated pepsin and found to be also
a protein.
-Haldane wrote a treatise entitled ‘Enzyme’ and made
remarkable suggestion about the interaction between enzyme
and substrate.
-In the twentieth century, research on enzymes has been
intensive in purification of thousand enzymes leading to
elucidate the structure and chemical mechanism.
Enzymes

-All enzymes are proteins, with exception of some RNA
molecules.
-Catalytic RNA molecules are called ‘ribozyme’ (self-splicing
intron and spliceosome, protein-RNA complex)
-Some antibodies can catalyze reaction called ‘abzymes’.

Enzymes

Function without Function with
additional chemical additional chemical
Components components
(only proteins)
Enzymes
Additional chemical components for enzyme function

Cofactors Coenzymes
One or more metal ions Complex organic or
-Fe2+ metalloorganic molecules
-Mg2+ -NAD+
-Mn2+ -PLP
-Zn2+ -Lipoate
-FAD
-Some enzymes require both cofactors and coenzymes.
-Some enzymes require either cofactor or coenzyme.
-Some enzymes contain many kinds of either cofactors or
coenzymes.
Enzymes
Holoenzyme = Apoenzyme + Cofactor or Coenzymes

Coenzymes or cofactors
covalently linked to enzymes
arecalled prosthetic groups.
Classification of Enzymes
Many enzymes have been named by adding the suffix ‘-ase’.
Enzyme Catalysis of Reactions

-Enhancement of the reaction rate is important to living system.

-Enzyme can catalyze reaction by providing a specific
environment within which a given reaction can occur more
rapidly.

-An enzyme-catalyzed reaction take places at the defined areas
called active site.

-The molecules that can bind to the active site are called
substrate.

-The active site of enzyme is lined with amino acid residues
with side chains that can bind the substrate and catalyze its
reaction.
Enzyme Catalysis of Reactions

-The function of enzymes is a catalyst by affecting on the rate
of reaction but they do not affect on chemical equilibrium.
-The effect of enzyme catalysis is an decrease in activation
energy (∆G#).
-Transition state is the
highest energy state of
compound undergoing
to product or returning
to substrate.
Enzyme Catalysis of Reactions
Principles of enzyme in lowering activation energy
-The binding energy (∆GB) between enzyme and substrate is
released from weak interactions to lower activation
energy (∆G#).
-The weak interactions are optimized substrate in transition
state: active sites of enzyme are complementary to transition
intermediate of substrate facilitating the conversion to product.

∆Gcat = ∆G − ∆GB
#
Weak interaction between enzyme and substrate in
transition state
Enzyme Kinetics
-Enzyme kinetic is the study of enzyme action.
-Enzyme action: chemical mechanism of an enzyme catalyze
reaction, determining the rate of reaction and responses to
changes experimental parameters, substrate concentration,
inhibition, temperature and pH.

Methods of measuring enzyme activity

-Monitoring enzyme activity = Enzyme assay
-Selection of a suitable physical and chemical technique for
following the appearance of product or disappearance of
substrate by widely used techniques, optical properties
(light absorption or fluorescence).
Methods of Measuring Enzyme Activity

Lactate Dehydrogenase
OH O O O
H3C C C O- + NAD+ H3C C C O- + NADH+
H

Lactate Pyruvate

d [ S ] d [ P] A340
r=− =
dt dt
Time
-[S]: lactate or NAD+ has no absorption in visible range.
-[P]: NADH has absorption at 340 nm.
-It is possible to monitor the reaction by assay at 340 nm
for product formation.
Approximation of Reaction Rate
-Substrate concentration is not constant at a time during
enzyme reaction.
The measurement of reaction rate
A0 at the initial period can be approximated
At = A0 e − kt that the substrate is not changed
At significantly when it is compared to the
starting concentration.
time
dA
-At the initial time At ~ A0 r=− = k[ At ]
dt
= k[ A0 ]
-The velocity of initial period is dependent on initial
concentration of substrate called initial velocity.
-Initial velocity is useful to measure the rate at defined
concentration of substrate.
Time Course of Reaction

Concentration Unit
[P] S P

[S]

Time
The time course reaction will be curved; as the concentration
of substrates decrease, and products accumulate, the net
forward rate decreases reaching to equilibrium.
Concentration

Equilibrium state
Slope of the tangent at time zero is
initial rate.

Time
Initial Rate
-Tangents to an experimentally obtained curve are not easy to
draw accurately.
-The suitable condition is to minimize the curvature by
decreasing the concentration of enzyme.

Low [E]: more accurate initial
velocity
Concentration

Enzyme activity 1 unit = A mount of
enzyme which converts one µ mole
of substrate per minute.

High [E]

Time
One-substrate Kinetics
The Michaelis-Menten equation
(Rapid-equilibrium assumption)

I. Concentration of [E]<<[S], therefore the all enzyme
molecules are in [ES] complex and [S]~[S0].
II. The initial rate is not affected by reverse reaction from
product accumulation because the initial period the product
is not significant.
III. The binding and dissociation of enzyme to substrate is
very fast (rapid-equilibrium assumption), so the rate-limiting
step becomes the product-releasing step.
One-substrate Kinetics
(Rapid-equilibrium assumption)
k1 k3
E+S ES E+P
k2 k4

At initial period, the product formation is not significant (k4~0).
k1 k3
E+S ES E+P
k2
e = [ E ] + [ ES ]
Rapid-equilibrium assumption k 2 >> k3

k1[ E ][ S ] = k 2 [ ES ]
Because the rate-limiting step is product releasing-step.
v = k3 [ ES ]
e = [ E ] + [ ES ] e = total enzyme concentration
One-substrate Kinetics
(Rapid-equilibrium assumption)

k3e[ S ] Vmax [ S ]
v= v=
[ S ] + k 2 / k1 [S ] + K m

k2
Michaelis-Menten constant Km =
k1
Maximum velocity Vmax = k3 [e]

Km is also referred to dissociation constant (Kd = k2/k1)
One-substrate Kinetics
Steady-state assumption
-In 1926, Briggs and Haldane showed that
the rapid-equilibrium is not restrictive in all
enzyme mechanism.
-They assume instead a steady
state that the concentrations
of E and ES remained
constant over the period of the
rate measurement.
One-substrate Kinetics
Steady-state assumption
k1 k3
E+S ES E+P
k2
d [ ES ]
The steady-state treatment =0
dt
Rate formation of ES

Rate decay of ES k 2 [ ES ] + k3 [ ES ]

k1[ E ][ S ] = (k 2 + k3 )[ ES ]
(k 2 + k3 )
[ E ] = [ ES ]
k1[ S ]
One-substrate Kinetics
Steady-state assumption
Because the rate-limiting step is product releasing-step.
v = k3 [ ES ]
e = [ E ] + [ ES ] e = total enzyme concentration

k3e[ S ] Vmax [ S ]
v= v=
k 2 + k3 [S ] + K m
[S ] +
k1
k 2 + k3
Michaelis-Menten constant Km =
k1
Maximum velocity Vmax = k3 [e]
Comparison between Rapid-equilibrium and
Steady-state Assumption
k1 k3
E+S ES E+P
k2

k 2 >> k3
k2
Rapid-equilibrium Km =
assumption k1
k 2 + k3
Steady-state Not restrictive Km =
assumption k1

-In case of rapid-equilibrium, k2 >> k3, k2 + k3 ~ k2, so
Km ≅ k2/k1.
-Michaelis-Menten assumption is only one case of steady-state
assumption.
Experimental Basis: Michealis-Menten Equation
-The relationship between initial rate and substrate concentration
is always hyperbolically depenent.

At very low substrate
[ S ] << K m ; K m + [ S ] ~ K m
Vmax [ S ]
Vmax [ S ] v=
v= Km
K m + [S ]
At substrate saturation
Vmax = k cat e
The Meaning of Kinetic Parameters

I. kcat: catalytic constant (turnover number)
-The value of kcat is nearly to the rate-limiting step.

k1 k3 k5
E+S ES EP E+P
k2 k4

fast fast slow

-The overall rate in enzyme-catalyzed reaction is not faster
than step of kcat (k5).

v = k cat [EP ]
-At saturated concentration
of substrate ([EP] = [E]total). Vmax = kcat [ E ]total
The Meaning of Kinetic Parameters

I. kcat: catalytic constant (turnover number)

-kcat is also referred to the number of cycles that enzyme
can catalyze the reaction.
-The reciprocal of kcat is referred to the time the enzyme
used since it binds to substrate until it releases of
product becoming free enzyme.
The Meaning of Kinetic Parameters

II. Km: Michaelis-Menten constant

-In all case, Km is the substrate concentration at which
Vmax
v=
2
-In case of rapid equilibrium that k2 is much more than other
steps, the Km can be referred to dissociation constant.
k2
Km =
k1
-In case of steady state assumption that k2 is not necessary
to be much more than other steps, the Km is a complex of
many rate constants. k +k
Km = 2 3
k1
Enzyme inhibition

-Inhibitors: some substances alter the activity of an enzyme
by combining with it influencing on substrate binding or its
turnover number in reducing enzyme activity.
-Most inhibitors are structurally resemble substrate but not
react or react slowly to enzyme.

Types of inhibitor
I Reversible inhibitors
-Inhibitors can bind and dissociate from enzyme active site
II Irreversible inhibitors
-Inhibitors that can not be removed from enzyme after binding
to enzyme
Competitive inhibition

E+S E:S E:P E+P
+
I
-Substances that can compete directly with a normal
substrate for an enzymatic binding site.
-A competitive inhibitor acts by reducing the
concentration of free enzyme available for substrate
E:I binding.
Derivations
k1 k3
E+S E:S E+P
+ k-1
I Rapid equilibrium for inhibitor binding

k-2 k2 KI =
[ E ][ I ]
[ EI ]
E:I [ E ]T = [ E ] + [ EI ] + [ ES ]

Initial rate (v) v = k3 [ ES ]

Maximal velocity (Vmax) Vmax = k3 [ E ]T
Derivations
k1 k3
E+S E:S E+P
+ k-1
I Define [E] and [EI] in term of [ES]

Steady-state assumption
k-2 k2
k1[ E ][ S ] = (k −1 + k3 )[ ES ]
E:I
Available of [E] in term of [ES]

[ E ][ I ]
KI = Substitution of [E] into KI
[ EI ]
Available of [EI] in term of [ES]
[ E ]T = [ E ] + [ EI ] + [ ES ]
Derivations
k1 k3
E+S E:S E+P
+ k-1
I
Initial rate (v) v = k3 [ ES ]
k-2 k2 Vmax = k3 [ E ]T

E:I

k3 [ E ]T [ S ] Vmax [ S ]
v= v=
[I ] [I ]
K m (1 + ) + [S ] K m (1 + ) + [S ]
KI KI
Analysis of the kinetic parameters
Primary plot; reciprocal plot of v versus [S]

1 Vmax [ S ]
v=
[I ]
v K m (1 + ) + [S ]
KI

1 Km [I ] 1 1
= (1 + ) +
v Vmax K I [ S ] Vmax

1
[S ] Km [I ]
Slope = (1 + )
Vmax KI
Slope varies depending on
inhibitor concentration. 1
Intercept =
Vmax
Analysis of the kinetic parameters
Secondary plot; Plot of any term functioning with
inhibitor concentration versus inhibitor concentrations

Km [I ]
Slope = (1 + )
Secondary plot Vmax KI

Slope
Km
Slope =
Vmax K I
Km
Intercept =
Vmax

[I]
Uncompetitive inhibition

E+S E:S E:P E+P
+
I

E:S:I

-Uncompetitive inhibitors bind only enzyme-substrate
complex and not to free enzyme
-Substrate binding could cause an enzyme conformational
change suitable for inhibitor binding.
Uncompetitive inhibition
1 K 1 [I ] 1
Primary plot =( m ) + (1 + )
v Vmax [ S ] K I Vmax
1 Km
v Slope =
Vmax

[I ] 1
Intercept = (1 + )
K I Vmax

1
[S ] Intercept is dependent on
inhibitor concentration.
Uncompetitive inhibition

[I ] 1
Intercept = (1 + )
K I Vmax
Secondary plot

Intercept 1
Slope =
K IVmax
1
Intercept =
Vmax

[I]
Non-competitive inhibition

E+S E:S E:P E+P
+ +
I I

KI for E ≅ KI for ES

E:I E:S:I

-Non-competitive inhibitor can combine with both enzyme
Form, ES or E molecule to produce dead-end complex.
-KI for E ≠ KI for ES, mixed inhibition
Non-competitive inhibition
1 Km [I ] 1 [I ] 1
= (1 + ) + (1 + )
Primary plot v Vmax K I [S ] K I Vmax

1
Km [I ]
v Slope = (1 + )
Vmax KI

Intercept = (1 +
[ I ] 1
)
K I Vmax
1
[S ]
Both slope and intercept vary depending on
inhibitor concentration.
Non-competitive inhibition
Secondary plot
1
Km
Slope Vmax K I Intercept K IVmax

Km 1
Vmax Vmax

[I] [I]

Km [I ] [I ] 1
Slope = (1 + ) Intercept = (1 + )
Vmax KI K I Vmax
Effect of inhibitor on kinetic parameters

Vmax [ S ]
Competitive inhibitor v=
[I ]
K m (1 + )
KI
Vmax
[S ]
[I ]
(1 + )
KI
v= Uncompetitive inhibitor
Km
+ [S ]
[I ]
(1 + )
KI Vmax
[S ]
[I ]
(1 + )
KI
Noncompetitive inhibitor v=
K m + [S ]
Two-substrate Kinetics
Enz
A+B P+Q

-For one substrate reaction, there is no argument about the
order of addition of reactants.
-For more than one substrate, there are possibilities of
order of substrate in a reaction.
I. Ternary complex (Sequential mechanism)
-Reaction can not be occurred until both substrates are
bound at enzyme active site.

A+B+E EAB P+Q
Two-substrate Kinetics

I. Ternary complex (Sequential mechanism)

Compulsory-order mechanism

E EA EAB EPQ EP E

- Substrate B can not bind to free enzyme, but only to binary
complex of EA.

Random-order mechanism
EA EP
-An either pathway in
binding substrate A and B
E EAB EPQ E
to enzyme forming a
central complex (EAB).
EB EQ
Two-substrate Kinetics

II. Ping-Pong mechanism
-Substrate A and B do not meet at the enzyme active site,
no EAB complex.
-One substrate must leave something on the enzyme to be
passed on to the other substrate.
A P B Q

E EA E' E'B E

-E′ can be referred to reducing equivalent, chemical group
such as phosphate group, acyl group and amino group.
(enzyme substitution mechanism or double displacement
mechanism)
Two-substrate Kinetics

I. Ternary complex (Sequential mechanism)
Reaction of enzyme alcohol dehydrogenase requires
ternary complex.

Alcohol:NAD+:Enz Acetalehyde:NADH:Enz

II. Ping-Pong mechanism

Reaction of enzyme glucose oxidase. Enzyme is in either
reduced and oxidized form during catalysis.

Glucose + E-FAD 1-ketoglucose + E-FADH2

E-FADH2 + O2 E-FAD + H2O2
Two-substrate Kinetics
General equation of two-substrate kinetics (Dalziel′s equation)

I. Ternary complex (Sequential mechanism)
e φ A φB φ AB
= φ0 + + +
v [ A] [ B ] [ A][ B ]
II. Ping-Pong mechanism
e φ A φB
= φ0 + +
v [ A] [ B]
e
At high concentration of A and B = φ0 Vmax = kcat e
Vmax
−1
kcat = φ0
Kinetic study of Two-substrate Reaction
-Determining of initial velocity (v) by fixing one concentration
of substrate [B] with varied concentrations of [A]
-Determining the reaction mechanism by primary plot, e/v
versus reciprocal values of substrate concentrations
-Determining of all Dalziel′s parameters from primary and
secondary plot

[B] [B]
e/v e/v [B]
[B] [B]
[B] [B]
[B]

1/[A] 1/[A]
Ternary complex
(Sequential mechanism) Ping-Pong mechanism