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Lecture 11
Enzymes: Kinetics
Key Concepts
Kinetics is the study of reaction rates (velocities).
Study of enzyme kinetics is useful for measuring
concentration of an enzyme in a mixture (by its catalytic activity),
its purity (specific activity),
its catalytic efficiency and/or specificity for different substrates
comparison of different forms of the same enzyme in different tissues or
organisms,
effects of inhibitors (which can give information about catalytic
mechanism, structure of active site, potential therapeutic agents...)
Dependence of velocity on [substrate] is described for many enzymes by the
Michaelis-Menten equation:
kinetic parameters:
Km (the Michaelis constant)
kcat (the turnover number, which relates Vmax, the maximum velocity,
to [Et], the total active site concentration)
kcat/Km (the catalytic efficiency of the enzyme)
can't be greater than limit imposed by diffusion control, ~108-109 M1sec1
Kinetic parameters can be determined graphically by measuring velocity of
enzyme-catalyzed reaction at different concentrations of substrate
(Vo vs. [substrate]).
Learning Objectives
Terminology: active site, enzyme-substrate complex, induced fit, initial
velocity, steady state, Vmax , Km , kcat , turnover number, KES , enzyme
efficiency.
Write out a simple Michaelis-Menten kinetic mechanism for an enzymecatalyzed reaction.
Recognize the Michaelis-Menten equation, and sketch a graph of Vo vs. [S]
for an enzyme-catalyzed reaction that illustrates Vmax and Km.
Define Km in terms of the rate constants in the Michaelis-Menten kinetic
mechanism; give the operational definition of Km that holds no matter
what the actual kinetic mechanism is for a particular enzyme.
Explain the relationship of kcat to Vmax, and the relationship of Km to KES.
State the units of Km, kcat, and Vmax.
Express the ratio of occupied active sites to total enzyme active sites
([ES]/[ET]) in terms of Vo and Vmax. What is the maximum possible value of
that ratio?
Given a plot of Vo/Vmax vs. [S], find the value of Km from the plot.
What two things is the parameter kcat/ Km used to indicate?
What sets the upper limit for the value of kcat/Km for an enzyme?
What is the approximate range of numerical values for that upper limit of
kcat/Km, with units?
Each reaction step has its own transition state with its own activation
energy (G).
If all of the individual steps' Gs are lower than the activation energy
of the uncatalyzed reaction, the overall rate of product formation will be
greater in the presence of the catalyst.
The overall rate of the catalyzed reaction is dictated by the slowest step
in a multistep reaction. Given a free energy diagram like the one in
Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed. (2004)
Fig. 6-3 (previous lecture notes), how do you identify the rate-limiting
(slowest) step on the reaction coordinate?
Active site:
relatively small part of whole enzyme structure
3-dimensional cleft with participating components from different parts
of primary structure
water often excluded so substrates and intermediates are in
non-aqueous environment (unless H2O is a reactant)
Berg et al.,
Fig. 8-7
Specificity of binding
depends on active site crevice being sterically and chemically
complementary to groups it is binding (best complementarity may be
present in ES complex but NOT in free enzyme -- induced fit)
Enzymes flexible -- conformational changes can occur when substrate
binds during the reaction, to get maximal complementarity to the
transition state.
induced fit: conformational changes giving tighter binding in a new
conformation
For many (probably most)
enzymes, the active site
assumes shape
complementary to S
only when S is bound.
Why we measure initial velocity, Vo, the slope of [P] vs. [time] at
very early time after mixing enzyme with substrate
Problem:
As S is converted to P, concentration of S decreases, so forward
velocity gets slower and slower.
Furthermore, as [P] increases, rate of reverse reaction (P --> S)
becomes significant, and eventually, when VF = VR, reaction is at
equilibrium: d[P]/dt = 0.
Solution:
Measure V at very early
times in reaction, before
[S] decreases signficantly
(so [P] = ~0).
Velocity measured
immediately after mixing
E + S, at beginning of
reaction (initial velocity),
is called Vo.
Meaning of Km
By definition,
Km = substrate concentration at which velocity (V) is exactly 1/2 of Vmax
(operational definition that holds for ANY kinetic mechanism).
Km is a SUBSTRATE CONCENTRATION.
Compare:
3.
Michaelis-Menten Equation:
kcat/Km
is the criterion of substrate specificity, catalytic efficiency and
"kinetic perfection.
units of kcat/Km = conc1time1.
k1 sets an upper limit on value of kcat/Km.
kcat/Km can never be greater than k1 (rate constant for association
of E + S) for a given enzyme-substrate system. (You're not
responsible for the explanation of this.)
How fast 2 molecules can react (or bind in the case of E + S) is
limited by how fast the molecules can diffuse to "bump into each
other" in solution so they can react.
For molecules the size of an enzyme and a typical substrate, the
maximum (diffusion-limited) k1 = ~ 108109 M1sec1.
Thus max. possible kcat/Km for an enzyme = ~ 108109 M1sec1.
Any enzyme with kcat/Km value in that range approaches the limit of
diffusion control, and thus has achieved something very close to "kinetic
perfection": the rate at which that enzyme's active site can convert
substrate into product is limited only by the rate at which it encounters
the substrate in solution.
kcat/Km
Uses of kcat/Km
kcat/Km used as a measure of 2 things:
1.
enzyme's substrate preference
2.
enzyme's catalytic efficiency
1. enzyme's preference for different substrates (substrate specificity)
The higher the kcat/Km, the better the enzyme works on that substrate.
e.g., chymotrypsin: protease that clearly "prefers" to cleave after bulky
hydrophobic and aromatic side chains.
chymotrypsin specificity:
active site best
accommodates
substrates with a
bulky hydrophobic
or aromatic residue
contributing carbonyl
group to peptide bond
to be hydrolyzed.
Berg et al., 5th ed., Fig. 9.1
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If KM = KES, which enzyme above binds its substrate the most tightly?
Which enzyme has the most rapid catalytic turnover when the enzyme
is saturated with substrate?
Which enzymes have the highest catalytic efficiency?
Are they near the limit of diffusion control?
NOTE: kcat/KM for an enzyme can have a value close to the limit of diffusion
control either because its kcat is very high, or because its Km is very low, or
some combination.
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Appendix:
How was the Michaelis-Menten Equation Derived?
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