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Department of Biochemistry
History of Enzymes
-Enzymes are biological catalysts.
-Each enzyme has a genetically
determined and unique primary
sequences.
Enzymes
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
-The molecules that can bind to the active site are called
substrate.
∆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.
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.
High [E]
Time
One-substrate Kinetics
The Michaelis-Menten equation
(Rapid-equilibrium assumption)
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]
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
k1 k3 k5
E+S ES EP E+P
k2 k4
v = k cat [EP ]
-At saturated concentration
of substrate ([EP] = [E]total). Vmax = kcat [ E ]total
The Meaning of Kinetic Parameters
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
k-2 k2 KI =
[ E ][ I ]
[ EI ]
E:I [ E ]T = [ E ] + [ EI ] + [ 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:I
[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
KI for E ≅ KI for ES
E:I E:S:I
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
Compulsory-order mechanism
E EA EAB EPQ EP E
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
E EA E' E'B E
Alcohol:NAD+:Enz Acetalehyde:NADH:Enz
[B] [B]
e/v e/v [B]
[B] [B]
[B] [B]
[B]
1/[A] 1/[A]
Ternary complex
(Sequential mechanism) Ping-Pong mechanism