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BIOC 460, Spring 2008

Lecture 11
Enzymes: Kinetics

Reading: Berg, Tymoczko & Stryer, 6th ed., Chapter 8,


pp. 216-225

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]).

LEC 11, Enzymes - Kinetics

BIOC 460, Spring 2008

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?

REVIEW: How do enzymes reduce activation energy (G)?


1. by lowering the free energy of the transition state (), e.g., by
binding the transition state tightly
2. by changing the reaction pathway by which reactants react to
form products; e.g., taking a 1-step uncatalyzed reaction and
accomplishing the same result by a different route, with several
intermediate reactions en route.

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?

LEC 11, Enzymes - Kinetics

BIOC 460, Spring 2008

BINDING = the essence of enzyme action!

binding of SUBSTRATE to form an ES COMPLEX


binding of TRANSITION STATE more tightly than the substrate
Binding occurs at ACTIVE SITE of enzyme.
Subsequent chemical events can then occur.

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)

Binding uses multiple weak interactions:


1. hydrogen bonds
2. salt links
3. van der Waals interactions
4. hydrophobic effect

Lysozyme (residues from different parts of AA sequence


come together in active site)

Berg et al.,
Fig. 8-7

LEC 11, Enzymes - Kinetics

BIOC 460, Spring 2008

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.

Berg et al., Fig. 8-10

Why study enzyme kinetics (reaction rates)?


measurement of velocity = reaction rate
compare enzymes under different conditions, or from different
tissues or organisms
understand how differences relate to physiology/function of organism
e.g., physiological reason for different Km values for hexokinase vs.
glucokinase (discussed later in course)
compare activity of same enzyme with different substrates (understand
specificity)
measure amount or concentration of one enzyme in a mixture by its
activity
measure enzyme purity (specific activity = amount of activity/amount of
protein)
study/distinguish different types of inhibitors
info about enzyme active sites and reaction mechanism
development of specific drugs (enzyme inhibitors)

LEC 11, Enzymes - Kinetics

BIOC 460, Spring 2008

Simple Enzyme-Catalyzed Reaction:


Measurement of velocity:
V = rate of appearance of product = change in [P] per unit time
units of velocity (V)? _______________

Plot of [P] vs. time


Experimentally,
forward velocity
VF = slope of plot of
[P] vs. time because
VF = kF[S] = [P]/t

Berg et al., Fig. 8.11


(slightly modified)

Determining initial velocity, Vo

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.

LEC 11, Enzymes - Kinetics

BIOC 460, Spring 2008

Vo vs. [S] plots


Simple uncatalyzed S P reaction
shows linear dependence of Vo on [S]:
Whats the slope of plot of VF vs. [S]?
Enzyme-catalyzed reactions
show a hyperbolic dependence
of Vo on [S]:
1. Rate is saturable: there's a
maximum rate (velocity = Vmax)
for any single concentration of
enzyme.
2. Rate (velocity) is proportional
to [Etotal]: at any single
concentration of [S], Vo = c[E]
(double [E] double Vo).
3. Half-maximal velocity occurs at a
specific substrate concentration,
independent of [E].
Km = substrate concentration
that gives Vo = 1/2 Vmax.

Dependence of Vo on [S] in enzyme-catalyzed reaction:

Plot of Vo vs. [S]) for an enzyme that follows Michaelis-Menten kinetics


plots as a rectangular hyperbola
approaches Vmax (= maximum velocity) at high [S]
3 parts of [S] concentration range
1. At very low [S]:
Vo is proportional to [S];
doubling [S] double Vo.
2. In mid-range of [S], Vo is
increasing less as [S]
increases (where Vo is
around 1/2 Vmax).
Km = [S] that gives
Vo = 1/2 Vmax.
3. At very high [S], Vo is
independent of [S]:
Vo = Vmax.

Berg et al., Fig. 8.12

LEC 11, Enzymes - Kinetics

BIOC 460, Spring 2008

Michaelis-Menten model to explain hyperbolic


dependence of Vo on [S] in enzyme catalyzed reactions
Before the chemistry occurs, enzyme binds substrate to
make a noncovalent ES complex.

Turnover number (def.): number of substrate molecules


converted into product by one molecule of enzyme active
site per unit time, when enzyme is fully saturated with
substrate. = k2 in M-M scheme above = kcat (general term)
kcat = turnover number (general term used, independent of
specific kinetic mechanism)
NOTE:
kcat (the turnover number) = k2 in the specific
Michaelis-Menten kinetic mechanism above.

The Michaelis-Menten Equation describes a rectangular hyperbola.

The Michaelis-Menten Equation

where Vmax = k2[ET] (so Vmax is indeed proportional to [ET])


Km: an "aggregate" constant (sum of rate constants for breakdown of ES
divided by rate constant for formation of ES):

Michaelis-Menten equation explains hyperbolic Vo vs. [S] curve:


1. At very low [S] ([S] << Km), Vo approaches (Vmax/Km)[S].
Vmax and Km are constants, so linear relationship between Vo and [S]
at low [S].
2. When [S] = Km, Vo = 1/2 Vmax
3. At very high [S], ([S] >> Km), Vo approaches Vmax (velocity
independent of [S])

LEC 11, Enzymes - Kinetics

BIOC 460, Spring 2008

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.

What is the equilibrium dissociation constant for ES complex (KES)?

Compare:

Km = KES (dissoc. constant for ES complex) only if k2 << k1.

Kinetic parameters used to characterize enzyme activity


Kinetic parameters useful for many comparisons, e.g., effects of enzyme
inhibitors on Km and Vmax = basis of diagnosis of type of inhibition.
effects immediately apparent on a double reciprocal plot (see
Enzyme Inhibition notes)
1. Km
(units: concentration units, e.g., M or mM or M)
IF k2 << k1 (not always true), then
Km = k-1/k1 = KES and
Km can be taken as a measure of the dissociation constant for the ES
complex (an INVERSE measure of the binding affinity of enzyme for that
substrate).
2.

kcat (directly related to Vmax) (units: inverse time, e.g., s1)


calculate kcat from Vmax and concentration of enzyme active sites [ET]:
Vmax = k2[ET] = kcat[ET] so

3.

kcat/ Km (the criterion of catalytic efficiency and "kinetic perfection")


(units: inverse conc.inverse time, e.g., M1s1)

LEC 11, Enzymes - Kinetics

BIOC 460, Spring 2008

Wide range of catalytic rate constants, kcat (turnover numbers) among


enzymes:
Lysozyme: kcat = 0.5 s1
Catalase: kcat = 4 x 107 s1
In vivo, most enzymes operate below saturation (V < Vmax)
(important for regulation)
Activity of some metabolic enzymes is regulated: V can be "tuned" up or
down depending on cell's metabolic needs.
Remember:
Vo = kcat[ES] = rate of appearance of P, and
Vmax = kcat[ET] so
(divide 1st equation by the second):
= fractional saturation!
Ratio of actual velocity at a given substrate concentration (Vo) to Vmax
indicates ratio of occupied active sites to total active sites at that [S].

Michaelis-Menten Equation:

Divide both sides by Vmax:

Can calculate fractional saturation


{ratio of (occupied sites / total active sites)} as

What is the maximum possible value of [ES]/[ET]?


What would be the shape of a plot of Vo/Vmax vs. [S], and
how would you find Km on the plot?

LEC 11, Enzymes - Kinetics

BIOC 460, Spring 2008

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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Substrate preferences of chymotrypsin


Chymotrypsin also catalyzes hydrolysis of ESTER bonds whose
carboxylic acid component has a bulky, hydrophobic and/or aromatic R
group.
Amino acids listed in Berg et al. Table 8-6 (below) are the groups that
contribute the carbonyl group to the ester bond being hydrolyzed by the
enzyme.

Which amino acid is most preferred by chymotrypsin for contribution of


carbonyl group in ester bond to be cleaved, among substrates above?

Catalytic efficiency of enzymes


the higher the kcat/Km, the more "efficient" the enzyme
maximum possible kcat/Km dictated by the diffusion limit

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?

To derive Michaelis-Menten Equation (describe hyperbolic Vo vs. [S] plot):


Start with the following 3 equations/assumptions:
1.
Enzyme active site concentration: [ETotal] = [Efree] + [ES]
[total active sites] = [empty sites] + [occupied sites]
2.
Total concentration of [S] is high enough that [Sfree] = [Stotal]
i.e., an insignificant fraction of total [S] is bound even when all the
enzyme active sites are occupied.
3.
In the STEADY STATE, concentration of [ES] is constant
(the steady state assumption):
rate of formation of ES = rate of breakdown of ES
(breakdown rate = sum of rates of breakdown forward + backward)
(See last 2 slides, Appendix, if youre interested in where to start the
derivation, but youre not responsible for it.)

M-M Equation Derivation, Steady State Assumption


Changes in conc. of enzyme-catalyzed reaction participants with time:
"Steady state" is the part of the reaction period that
concentration of ES is not changing:
rate of formation of ES = rate of breakdown of ES
k1[E][S] = k1[ES] + k2[ES]

Berg et al., 5th ed.


Fig. 8-13a

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