BIO 3601 - Biochemistry

Enzyme Kinetics
Enzyme Kinetics
Enzyme Chemistry

ratio between the rate (speed) of the

catalyzed and uncatalyzed reaction
 Specific Questions

• What is the maximal rate the enzyme converts

substrates into products?

• How does changing the substrate

concentration affect the rate of catalysis?

• How does the enzyme “choose” its substrate?

How do we know which substrates are acted
on by specific enzymes?
 Chemical Kinetics
• The reaction mechanism is the exact molecular
pathway followed by starting materials in the
transition to products.

• Chemical kinetics is the study of the rates of

chemical reactions.

• Rate laws are mathematical models of chemical

mechanisms.

• Experimental measurements of chemical reaction

rates can be used to verify or refute specific rate
laws.
 Simple Kinetics

Blue  Green

t=0s t=1s t=2s t=3s

 Rate Expressions

-(D[blue]/Dt) = (D[green]/Dt) = kbg [blue]

rate constant
• The rate constant relates the concentration of
the substrate to the rate of the formation of
product.

• To determine the rate constant, we can

measure the effect of the concentration of blue
on the rate of formation of green.
Measurement of Reaction Rates (Kinetics)

Progress Curve (Raw Data) M-M Plot (Analyzed Data)

Product Concentration

Slope (DP/DT)
 Progress Curves

Question: why does the progress curve asymptote instead

of continuing in a linear way?
 Enzyme Kinetics of Invertase

Sucrose  glucose + fructose

Each point on this graph is a slope

from a progress curve recorded at
a given concentration of substrate.

What is the rate law for this reaction?

(Brown, 1904)
 Rate Law for Invertase

k1
E+S P at low substrate
concentration
k2
E P at high substrate
concentration

How can the rate law change

as a function of concentration?
 Brown-Henri Hypothesis
Enzymes (E) and their substrates (S) form a stable complex (ES) prior to
catalysis.
Low substrate
Enzyme
concentration
Increasing substrate
concentration
Substrate
increases rate of
product formation.

High substrate
concentration
Increasing substrate
concentration does
not result in an
increase in product
formation.
 “
Where have I seen this before?”
“What can I do with all this information”?
 Myoglobin binding

L
L L
L
 Rate Constants – ka and kd
Association rate = ka [P] [L]
Dissociation rate = kd [PL]

ka (M s )
-1 -1 kd (s-1)

At equilibrium, these two rates are equal.

P+L PL A kinetic view…..for

now!

Kd = [P][L]/[PL] = kd/ka
 Brown-Henri Hypothesis
This part looks just like myoglobin
binding to oxygen.
This is what we are
interested in knowing.

• This is the steady state assumption, that the enzyme-substrate

complex forms at the same rate that it falls apart.
• Also notice a difference between enzymes and myoglobin.
Myoglobin-oxygen has only one way to fall apart but the enzyme-
substrate complex has two ways to fall apart.
• To make this useful, solve for [ES]. This will yield the M-M equation.
 Henri-Michaelis-Menten Model

Initial rate/velocity VO of product formation dictated by second step:

Vo = k2[ES]
total enzyme

[S]
Vo = k2 [E]total
[S] + (k-1 + k2)/k1
rate (conc/time)
Deriving the Michaelis-Menten Equation
total enzyme

[S]
Vo = k2 [E]total
[S] + (k-1 + k2)/k1
rate (conc/time)

Recalling that Vo = k2[ES], Vmax = k2[E]total

Vo = Vmax [S]
[S] + Km
 Michaelis Constant Km
E E
k1 E k2
+
k-1 S
S P
First order reactions
(s-1)

Second order
reaction (M-1 s-1)

Km is a measure of substrate occupation of enzyme,

or the ability of the enzyme to capture substrate
 Michaelis Constant Km

V0 = Vmax [S]
[S] + Km

If v0 = ½ vmax, then …
Michaelis Constant Km
Michaelis Constant Km

Km is a property of the enzyme-substrate pair,

not an intrinsic property of the enzyme
 Biological Relevance of Km

Glucose is phosphorylated by both hexokinase (Km = 0.1 mM) and

glucokinase (Km = 5.0 mM). Because glucokinase expression is largely
restricted to the liver, the blood and muscles get preferential access to
glucose under fasting conditions.
 kcat and Vmax

Maximum velocity Turnover number, rate at which

one enzyme turns S to P at
saturation [S]

Substrate Enzyme
kcat
Michaelis-Menten Equation(s)

[S]
Vo = Vmax
[S] + Km

[S]
Vo = kcat [E]T
[S] + Km
 Michaelis-Menten Equation(s)
[S]
Vo = Vmax
[S] + Km

Vo [S]
=
Vmax [S] + Km

fraction of occupied
enzyme (ES)
fES
 Determining Kinetic Parameters

Lineweaver-Burk Plot
(“double” reciprocal plot)
 Saturation Kinetics and Vmax
Initial velocity, Vo (mM/min)
Vmax

How are these approximations

derived from the M-M equation?

Substrate concentration (mM)

 Kinetic Parameters and Specificity
• How does an enzyme “decide” which substrate to act on?

• How do we know what the “correct” substrate for an

enzyme is?

• If we consider Km, the smaller the value of Km, the more

efficient the enzyme captures substrate at a lower substrate
concentration.

• If we consider kcat, the higher the value of kcat, the faster the
enzyme converts substrate into product.

• Which is the correct parameter to choose in deciding which

substrate is preferentially catalyzed by a given enzyme,
particularly at low substrate concentrations?
 Catalytic Efficiency and Substrate
Specificity
At low substrate concentrations, [E] ~ [ET]. The ratio kcat/KM is a second-order rate constant for
the reaction between a substrate and free enzyme, providing a measurement of enzyme
specificity.

Based on these numbers, which substrate is preferred by this enzyme?

Copyright © 2013 Pearson Canada Inc.
 Low Substrate Conditions
Vmax

Vo
(mM/
First order
min) in [S] and [E]

Vo ≈ (kcat/Km) [ET] [S]

Substrate concentration (mM)

kcat/Km is a pseudo-second order rate constant

for the reaction [E] + [S]  P at low [S]
 Catalytic Perfection
kcat/Km = k2/((k-1 + k2)/k1) = k2k1/(k-1 + k2)

If k2 > k-1, every substrate binding step results in a

product: kcat/Km → k1 (diffusion-limited rate). This is
the state of catalytic perfection.
 Speed Limits
kcat/Km = k2/((k-1 + k2)/k1) = k2k1/(k-1 + k2) < k1
 Breaking the Speed Limits

The enzyme superoxide dismutase appears to catalyze a reaction faster than the rate
of diffusion. How can this happen? The substrate superoxide O2- is negatively charged
and small, while the active site (green area) is surrounded by positively charged side
chains. Thus, diffusion is not the only way the substrate finds itself in the active site.
How Do They Make These?