Enzymes Kinetics

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Enzymes Kinetics
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Learning Objectives
Write the Michaelis-Menten equation, and
rearrange it to form the Lineweaver-Burk equation.
Describe the assumptions in the derivation of the
Michaelis-Menten equation.
Construct a double-reciprocal plot from kinetic
data, and calculate Km and Vmax.
Describe the different types of reversible enzyme
inhibition, and recognize the characteristic
double-reciprocal plot of each.
Describe irreversible inhibition.
Describe an allosteric enzyme.
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enzyme kinetics a discipline wherein the
mechanism of an enzyme catalyzed reaction is
investigated by determining the rate of the
reaction and how it changes in response to
changes in experimental parameters.
substrate concentration, S
E

S
ES
P
E

An enzyme catalyzed reaction is started by adding
S to E. As the reaction progresses, S is used
up S decreases. Thereforemeasure the initial
velocity, Vo, or initial ratebefore the
substrate has decreased to any great extent.
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When the reaction is initiated S is much
greater than E changes in S are negligible
if the measurement of Vo is made quickly S
can be assumed to be constant over the short
time course of measurement of the velocity.
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Vmax maximal velocity
The amount of added enzyme is constant
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The enzyme combines with substrate in a
relatively fast, reversible step
k1
E

S
ES
k-1
ES then breaks down to product in a slower step
k2
ES
P
E

k-1 gtgt k2
ES can be assumed to be constant steady-state
assumption
Since the release of product is rate-limiting,
the overall rate of the reaction is proportional
to ES.
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Michaelis-Menten Equation
Operational definition of Km when S Km,
Vo 1/2 Vmax
Vmax S
At low S S ltlt Km
Vo
Km
At high S S gtgt Km
Vo Vmax
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Lineweaver-Burk Plot
(double-reciprocal plot)
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turnover number equivalent to the number of
substrate molecules converted to product in a
given unit of time
Vmax
Kcat
ET
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Reversible Inhibitors
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Competitive Inhibition
Km changes
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Uncompetitive Inhibition
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Mixed Inhibition
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Irreversible Inhibitors
Reaction of chymotrypsin with DIFP
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suicide inhibitors relatively unreactive until
they bind at the active site of a specific
enzyme it is then converted by the enzyme into a
very reactive compound that reacts irreversible
with the enzyme.
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Allosteric substrate-activity curve
homotropic allosteric enzyme substrate is a
positive activator.
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Chymotrypsin
Cleaves peptide bonds involving the carboxyl of
aromatic amino acids
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