5.

07 Lecture 6

Enzyme Kinetics

Unraveling metabolic pathways and their regulation

For the model reaction S P, the time course of product formation is shown
below.
Rates (v) of enzymatic and non-enzymatic reactions as a function of time,
for example, product concentration ([P]) versus time (t).

v = -d[S]/dt = d[P]/dt = k[S]

Initial rates. To simplify analysis, all our discussions of enzyme kinetics will be
limited to initial rates (vo; also referred to as initial velocities), that is, up to ~10%
of the substrate-to-product conversions. During this initial time period, [S] [S] o.

How do kinetics of enzymatic reactions differ from those

of uncatalyzed chemical reactions?

Initial rate of product formation (v o) vs. [S]o

For enzymatic reactions, the initial rate (v o) of the reaction is directly proportional to [S]o at low substrate co ncentrations, as it is for the non-catalyz ed reaction. How ever, at high [S]o, the enzymatic reaction, in contrast to an uncatalyzed one, becomes zero order in [S] o; i.e ., vo is independent of the [S]o. Since the active sites of the enzymes are fully saturated with S, further increasing its [S] o has no effect on the reactio n rate.

How can we mathematically describe these experimental observations?

Michaelis-Menten Equation
k-1

k2

E + S ES E + P
k1
We need a description of the overall rate of the reaction, i.e., the
concentration of the product formed per unit of time:
v = d[P]/dt = k2[ES]
In general, if a step is irreversible, then choosing this step to describe the
rate of product formation simplifies the algebra. The answer is the same
no matter what step we choose. The [ES] usually is not experimentally
measurable. One, therefore, needs to define [ES] in terms of
experimentally measurable parameters, namely [E]o and [S]o.
Since the enzyme-substrate complex ES is formed in one reaction and
decomposed in two, then
d[ES]/dt = k1[E][S]o k-1[ES] - k2[ES]
To integrate this differential expression with its three variables,
additional simplifications need to be made.

Michaelis-Menten Equation
k-1

k2

E + kS ES E + P
1

ES is a non-covalent enzyme-substrate complex

Conditions and assumptions:
1. In a typical experiment, [S]o >> [E]o:
is mM to M and the [E]o is nM to M.

the [S]o

2. The reaction is run under conditions were only a few % of substrate (under
~ 10%) is converted to product (initial velocity conditions). Therefore, v =
vo and [S] = [S]o
3. Substrate conservation equation: [S]o = [S] + [ES] [S]o

The [S]o can be experimentally measured (UV/Vis absorbance

spectroscopy if S has a chromophore or scintillation counting if the
substrate is made radioactive).
4. Enzyme conservation equation: [E]o = [E] + [ES]
The [E]o is experimentally measurable. Use the A280; see website

http://ca.expasy.org/ under Expasy proteomics tools, one can estimate the

extinction coefficient of a protein whose sequence is known.

Michaelis-Menten Equation
Steady-state assumption (generally valid under the conditions outlined
above and greatly facilitating solution to the problem):
d[ES]/dt = 0
Therefore, after a brief time period called the pre-steady state
(milliseconds, see lag phase below), the rate of formation of ES is equal to
the rate of its disappearance:
k1[E][S]o = k-1[ES] + k2[ES]

Michaelis-Menten Equation

Now rearrange the expression for [ES] in terms of experimentally

measurable parameters given that [E]o = [E] + [ES] :
[ES] = k1[E]o[S]o/(k-1 + k2 + k1[S]o)

Using the rate expression v = k2[ES] gives

vo = k1k2[E]o[S]o/(k-1 + k2 + k1[S]o)
where vo is experimentally measurable, [E]o and [S]o are known, and the
[S]o can be varied.

We thus have two known variables ([E]o and [S]o) and three unknown rate
constants (k1, k-1, and k2). We cannot solve the equation for the
individual rate constants and need to regroup them into new constants,
Vmax and KM, as shown on the next two slides.

Michaelis-Menten Equation
Vmax
Vmax = k2[E]o = kcat [E]o
In this case, k2 (or, in general, kcat for more complex enzymatic reactions) is the

turnover number of the enzyme. It is the number of reaction processes that

each enzyme active site catalyzes per unit time.
kcats units are time-1, usually s-1. kcat is in general composed of a number of first
order rate constants that cannot be evaluated individually in the steady-state
analysis.
Attaining Vmax implies that all the enzyme active sites are tied up with the

substrate and, therefore, the reaction cannot go any faster under given
conditions (e.g., of pH and temperature).

Michaelis-Menten Equation
KM = (k-1 + k2)/k1
KM (Michaelis-Menten constant; alternatively denoted as K m) is the [S]o at which
the vo reaches 1/2 Vmax.
The units of KM are those of concentration, usually M or mM.
If k-1 >> k2, then KM = k-1/k1 = Kd; Kd is the thermodynamic dissociation constant
reflecting the affinity of the substrate to the enzyme. In most cases,
however,the KM is not the measured thermodynamic affinity of the E for the S;
rather, as with kcat, it is also composed of a number of rate constants.
KM is unique for each given enzyme pair under a particular set of conditions (such
as pH and temperature). The smaller the KM value, the lower is the substrate
concentration at which the enzymatic reaction rate reaches the V max, that is, the
maximal catalytic efficiency under given conditions (e.g., of pH and temperature).

Michaelis-Menten Equation

Substituting these new constants into the above equation

vo = k1k2[E]o[S]o/(k-1 + k2 + k1[S]o) gives the Michaelis-Menten equation:
vo = Vmax[S]o/(KM + [S]o) or

kcat and kcat/KM are the two most important enzyme/substrate kinetic
parameters.

kcat: turnover number, tells us how good our enzyme is as a catalyst.

kcat/KM: proficiency constant or specificity constant, tells us how near to

perfection our enzyme has evolved as a catalyst and how well it likes one
substrate over another.

Why are these the important kinetic parameters?

Michaelis-Menten kinetics
vo = Vmax[S]o/(KM + [S]o)

Initial rate of
product formation
(vo)
Initial substrate
concentration ([S]o)

Let us look at the two extreme scenarios:

1. If [S]o , then vo Vmax Reaction is zero-order in substrate. Tells us how
good our enzyme is as a catalyst.
2. If [S]o 0, then vo (k2/KM)[E]o[S]o. Under these conditions, the reaction is
first order in both substrate and enzyme. k2/KM is a second-order rate constant
called the specificity (or proficiency) constant. This constant is upper-limited by
diffusion, i.e., when the enzyme and the substrate need to find each other in
aqueous solution. The diffusion-controlled rate constant in enzymatic reactions is
typically between 106 and 109 M-1s-1. The physical step is rate-limiting, and the

Accounts of Chemical Research (2001) article by Wolfenden

kcat/KM values are relatively similar for enzymatic reactions as compared

to uncatalyzed reactions

Evaluation of kinetic parameters is based on fits

to Michaelis-Menten equation
vo = Vmax[S]o/(KM + [S]o)
Lineweaver-Burk plots provide a method, often used in enzyme
kinetics, to linearize the vo vs. [S]o data and quickly assess the Vmax
and KM values and visually present kinetic data:
1/vo = KM/Vmax(1/[S]o) + 1/Vmax

[ y = mx + b ]
Since Vmax= kcat[E]o, if [E]o is known,
then
kcat = Vmax/[E]o

Limitations of steady-state enzyme kinetics

By means of steady-state enzyme kinetics, the scheme
E + S ES E + P
cannot be distinguished from that where ES comprises
ES1 ES2 ES3 ESn
i.e., when there are several enzyme intermediates in equilibrium with each
other. Therefore, the steady-state kinetic analysis of an enzymecatalyzed reaction cannot unambiguously establish its mechanism (but
can rule out a mechanism whose predictions contradict experimentally
observed kinetic data).
Nor can the steady-state kinetic analysis of an enzyme-catalyzed
reaction determine k1, k-1, or k2 individually. Pre-steady-state methods
are required for that.
For such more complex reactions, the expressions for k cat and KM become
very complicated (denoted as kcat(app) and KM(app), respectively).
Importantly, however, the specificity constant k cat(app)/KM(app) still equals
kcat/KM.

Bisubstrate (or multisubstrate) enzyme reaction

kinetics
Most known enzymatic reactions involve two (rather than just one)
substrates and form two products. They are often group-transfer
reactions or redox reactions:
E
A X + B A + B X

For example, the enzyme alcohol dehydrogenase (ADH) catalyzes

oxidation of ethanol to acetaldehyde using NAD+ as an oxidizing agent
(i.e., ethanols hydride is formally transferred from ethanol to NAD +):
ADH
CH3CH2OH + NAD+ CH3CH(=O) + NADH

Such bi- (or multi-) substrate reactions typically occur via one of the
following two kinetic mechanisms: (i) sequential (single-displacement)
reactions, and (ii) ping-pong (double-displacement) reactions.

Bisubstrate (or multisubstrate) enzyme reaction kinetics

(i) Sequential (single-displacement) reactions

All substrates must combine with the enzyme before the reaction can occur and
products be released.
For the enzyme-catalyzed reaction A X + B, the X group being transferred is directly
passed from A to B. These sequential reactions may have an ordered mechanism, where
there is a compulsory order of substrate addition to the enzyme, or a random mechanism,
where there is no preference for the order of substrate addition to the enzyme.

In an ordered mechanism, the binding of the first substrate causes a conformational

change in the enzyme molecule which forms the binding site for the second substrate. In a
random mechanism, both binding sites pre-exist in the free enzyme. The just-considered
reaction
CH3CH2OH + NAD+ CH3C(=O)H + NADH

follows an ordered bisubstrate mechanism, in which NAD+ is the leading substrate.

Bisubstrate (or multisubstrate) enzyme reaction kinetics

(ii) Ping-pong (double-displacement) reactions
One or more products is/are released before all substrates have
combined with the enzyme.

In this case, for the enzyme-catalyzed reaction A X + B, the X group

being transferred is not directly passed from A to B: rather, it typically
covalently binds to the enzyme to form the enzyme intermediate E X,
while the first product A is released (Ping). In the second, Pong
stage, the X is transferred from the E X to the second substrate B
and the enzyme E is regenerated.
Bisubstrate enzyme-catalyzed reactions have two pairs of kcat(app) and
KM(app) (one pair for each substrate) and complicated Michaelis-Menten
equations. Importantly, however, the sequential and ping-pong
mechanisms can be distinguished from each other by steady-state
enzyme kinetic measurements.