BIOCHEMISTRY (I)
LIFS2210
Enzyme Kinetics (I)
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Enzyme Kinetics
Several terms to know!
• rate or velocity
• rate constant
• rate law
• order of a reaction
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Kinetic Experiments Reveal
Enzyme Properties
Chemical Kinetics
• Experiments examine the amount of product
(P) formed per unit of time ([P] / t)
• Velocity (v) - the rate of a reaction
(varies with reactant concentration)
• Rate constant (k) - indicates the speed or
efficiency of a reaction
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1. First order reactions
• For an irreversible reaction, A -> B, the reaction
rate (velocity, V) is given by
– for the rate of appearance of the product B
V = d[B]/dT
– for the rate of disappearance of the substrate A
V = -d[A]/dT
• These equations are equally valid for this
reaction, so we can say
V = d[B]/dT = -d[A]/dT = k1[A]
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First order reactions (cont)
• k1 is called the rate constant (k is expressed in
reciprocal time units (s-1))
• This type of a reaction is called a first-order
reaction, because its rate depends on the first
power of the reactant concentration.
• If k1 is large, the reaction is fast and if k1 is
small, the reaction is slow.
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First order reactions (cont)
Integrating the above equation yields
where [A]0 is the starting concentration of A (when t = 0)
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Determining the order of an irreversible first-
order reaction
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Determining the rate constant of an
irreversible first-order reaction
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Reversible reaction
k1
• For the reaction A B
k-1
• A forward reaction constant k1 can be used to define
the reaction moving rightward
• A reverse reaction constant k-1 can be used to define
the reaction moving leftward
• -V = d[A]/dt = -k1[A] + k-1[B]
• At equilibrium, 0 = -k1[A]eq + k-1[B]eq
• or [B]eq/[A]eq = k1/k-1 = Keq
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2. Second order reaction
• For reactions: S1 + S2 P1 + P2
• Rate is determined by the concentration
of both substrates
• Rate equation: v = k[S1]1[S2]1
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Second-Order Reactions (cont)
• A simple example is
2A A2
• The rate of such a reaction is proportional to
the second power of the concentration of the
reactant.
V = -d[A]/dt = k2[A]2
• k2 is the second-order rate constant.
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Pseudo first order reaction
• If the concentration of one reactant is so high
that it remains essentially constant, reaction
becomes zero order with respect to that reactant
• Overall reaction is then pseudo first-order
v = k[S1]1[S2]0 = k’[S1]1
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3. Enzyme Kinetics
• Enzyme-substrate complex (ES) - complex
formed when specific substrates fit into the enzyme
active site
E + S ES E+P
• When [S] >> [E], every enzyme binds a molecule
of substrate (enzyme is saturated with substrate)
• Under these conditions the rate depends only
upon [E], and the reaction is pseudo-first order
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Effect of enzyme concentration [E]
on velocity (v)
• Fixed, saturating [S]
• Pseudo-first order
enzyme-catalyzed
reaction
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Initial velocity (vo)
• Velocity at the beginning of an enzyme-catalyzed
reaction is vo (initial velocity)
• k1 and k-1 represent rapid noncovalent association
/dissociation of substrate from enzyme active site
• k2 = rate constant for formation of product from ES
k1 k2
E+S ES E+P
k-1
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Progress curve for an
enzyme-catalyzed reaction
• The initial velocity (vo)
is the slope of the initial
linear portion of the
curve
• Rate of the reaction
doubles when twice as
much enzyme is used
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Initial velocity (vo)
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The steady state of enzyme kinetics
• Steady-state conditions:
Rate of ES formation = Rate
of ES decomposition
k1 k2
E+S ES E+P
k-1
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4. The Michaelis-Menten Equation
• Louis Michaelis and Maude Menten's theory
• It assumes the formation of an enzyme-
substrate complex
• It assumes that the ES complex is in rapid
equilibrium with free enzyme
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The Michaelis-Menten Equation
• Maximum velocity (Vmax) is reached when an
enzyme is saturated with substrate (high [S])
• At high [S] the reaction rate is independent of
[S] (zero order with respect to S)
• At low [S] reaction is first order with respect to S
• The shape of a vo versus [S] curve is a
rectangular hyperbola, indicating saturation of
the enzyme active site as [S] increases
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Plots of initial velocity
(vo) versus [S]
(a) Each vo vs [S] point is
from one kinetic run
(b) Michaelis constant (Km)
equals the concentration
of substrate needed for
1/2 maximum velocity
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The Michaelis-Menten equation
• Equation describes vo versus [S] plots
• Km is the Michaelis constant
Vmax[S]
vo =
Km + [S]
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The Meanings of Km
• Km is a constant
• Km = [S] when vo = 1/2 Vmax
• Km can be a measure of the affinity of E for S
The lower the value of Km, the tighter the substrate
binding
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Km and physiological
substrate concentrations
• Km values for enzymes
are typically just above
[S], so that the reaction
rate is sensitive to small
changes in [S]
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Thinking about Km
• Km is unique for each and every
enzyme-substrate pair
• The Km value is also a function of pH
and temperature.
– Why?
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Understanding Vmax
The theoretical maximal velocity
• Vmax is a constant
• Vmax is the theoretical maximal rate of the
reaction - but it is NEVER achieved in reality
• To reach Vmax would require that ALL enzyme
molecules are tightly bound with substrate
• Vmax is asymptotically approached as
substrate is increased
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5. Kinetic Constants Indicate Enzyme
Activity and Specificity
• Catalytic constant (kcat) - first order rate
constant for conversion of ES complex to E + P
k1 k2
E+S ES E+P
k-1
When [S] >> [E], Vmax = k2[E]t or Vmax = kcat[E]t
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The turnover number
A measure of catalytic activity
• kcat, the turnover number, is the number of
substrate molecules converted to product per
enzyme molecule per unit of time, when E is
saturated with substrate.
• If the M-M model fits, k2 = kcat = Vmax/Et
• Values of kcat range from less than 1/sec to
many millions per sec
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to
When [S] << Km,
Ratio kcat/Km is a second order rate constant for
E+S E + P at low [S] concentrations
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Meanings of kcat and kcat/Km
•Km: binding affinity of E and S
•kcat: turnover rate of ES to E + P
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The catalytic efficiency
Name for kcat/Km
• An estimate of "how perfect" the enzyme is
• It measures how the enzyme performs
when S is low
• The upper limit for kcat/Km is the diffusion
limit - the rate at which E and S diffuse
together
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Values of kcat/Km
• kcat/Km can approach rate of encounter of two
uncharged molecules in solution (108 to 109M-1s-1)
• kcat/Km results in the rate constant that measures
catalytic efficiency.
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Preference of chymotrypsin in the hydrolysis of
several N-acetyl amino acid methyl esters
Hydrolysis efficiency as measured by kcat/KM
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kcat and Catalytic Efficiency
Therefore, enzymes that have high kcat/KM ratios have
essentially attained kinetic perfection because they
have come very close to reaching complete efficiency
only being limited by the rate at which they encounter
the substrate in solution.
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