Michaelis-Menten kinetics

Enzymes catalyze reactions in physiological systems. In an equilibrium, an enzyme (E)

binds a substrate (S) to form an enzyme-substrate complex (E-S). The E-S complex can
dissociate or irreversibly convert the substrate to a product (P) (Scheme 1). The
Michaelis-Menten equation describes the relationship between the rate of substrate
conversion by an enzyme to the concentration of the substrate (Equation 1). In this
equation, V is the rate of conversion, Vmax is the maximum rate of conversion, [S] is the
substrate concentration, and Km is the Michaelis constant. The Michaelis constant is
equivalent to the substrate concentration at which the rate of conversion is half of Vmax.
Km approximates the affinity of enzyme for the substrate. A small Km indicates high
affinity, and a substrate with a smaller Km will approach Vmax more quickly. Very high
[S] values are required to approach Vmax, which is reached only when [S] is high enough
to saturate the enzyme. While the derivation is not shown in this discussion, Vmax is
equivalent to the product of the catalyst rate constant (kcat) and the concentration of the
enzyme.

Scheme 1 - enzyme-substrate complex and product formation model

(1)
Applet

This applet plots V vs [S] data for up to three compounds/situations. For each
compound, Km and Vmax must be specified. The units of Km and [S] are concentration,
e.g. mM or µM. The units of Vmax and V are amount of product over time, typically
µmol/min or similar.

Substrate (+ Inhibitor) Vmax Km

(Color) (amount time-1) (conc.)
one (blue)
two (red)
three (green)
calculation may be slow
How km inhibit leukemia
pp 95 - 102

A new inhibitor of Bcr-abl kinase, a key therapeutic target for treating chronic
myelogenous leukemia (CML), is reported in the February issue of Nature Chemical
Biology. CML is a common form of leukemia that is typically characterized by two
genes, BCR and ABL1, connecting together when they should be separate. The
resulting fusion protein causes cellular signaling pathways to be on when they should
be off and contributes to the development of CML. CML patients are developing
resistance to the current clinical inhibitor of these signaling pathways, so there is a
strong need to find therapeutics with alternative mechanisms of action.

Nathanael Gray and colleagues screened for new Bcr-abl inhibitors by looking for
small molecules that were toxic to Bcr-abl�expressing cells but not normal cells. A
compound identified in the screen was shown to selectively inhibit leukemic cell lines
and use a different mechanism to bind to the protein from the current inhibitor. The
highly selective Bcr-abl inhibition and distinct mode of action suggest that this
inhibitor is a valuable lead for developing new CML treatments.

How to derived Michael-Menten

A simple model of enzyme action:

In this model, the substrate S reversibly associates with the enzyme E in a first step, and
some of the resulting complex ES is allowed to break down and yield the product P and
the free enzyme back. We would like to know how to recognize an enzyme that behaves
according to this model. One way is to look at the enzyme's kinetic behavior -- at how
substrate concentration affects its rate. So we want to know what rate law such an
enzyme would obey. If a newly discovered enzyme obeys that rate law, then we can
assume that it acts according to this model. Let's derive a rate law from this model.

For this model, let v0 be the initial velocity of the reaction. The latter stands for the
appearance of the product P in solution (+ d[P]/dt) whose phenomenological rate
equation (first-order) is given by

v0 = kcat[ES] --------(2),
containing an experimentally measurable (dependent) variable - v0, a kinetic parameter -
kcat, and another variable unknown to us - [ES].

Before proceeding, one should state (and remember) some implicite

assumptions:

• As long as initial velocity is considered, the concentration of product

can be neglected (compared to that of the substrate, thus [P] << [S]),
and

• The concentration of substrate is in large excess over that of the

enzyme ([E] << [S]).
These assumptions, which hold in most kinetic experiments performed in test
tubes at low enzyme concentration, are convenient when considering the
mass conservation equations for the reactants
[S]0 = [S]free + [ES] + [P] which now approximates to [S]0 = [S],
while that for the enzyme is
[E]total = [E]free + [ES] (the possible formation of a complex EP is not
considered here).

We want to express v0 in terms of measurable (experimentally defined, independent)

variables, like [S] and [E]total , so we can see how to test the mechanism by experiments in
kinetics. So we must replace the unknown [ES] in (2) with measurables.

During the initial phase of the reaction, as long as the reaction velocity remains constant,
the reaction is in a steady state, with ES being formed and consumed at the same rate.
During this phase, the rate of formation of [ES] (one second order kinetic step) equals its
rate of consumption (two first order kinetic steps). According to model (1),

Rate of formation of [ES] = k1[E][S].

Rate of consumption of [ES] = k-1[ES] + kcat [ES].

So in the steady state,

k-1[ES] + kcat[ES] = k1[E][S] (3)

Remember that we are trying to solve for [ES] in terms of measurables, so that we can
replace it in (2). First, collect the kinetic constants, and the concentrations (variables) in
(3):
 (k-1 + kcat) [ES] = k1 [E][S],
and (4)
(k-1 + kcat)/k1 = [E][S]/[ES]

To simplify (4), first group the kinetic constants by defining them as Km:

Km = (k-1 + kcat)/k1 (5)

and then express [E] in terms of [ES] and [E]total, to limit the number of unknowns:

[E] = [E]total - [ES] (6)

Substitute (5) and (6) into (4):

Km = ([E]total - [ES]) [S]/[ES] (7)

Solve (7) for [ES]:

First multiply both sides by [ES] (no Black Magic involvement here...):

[ES] Km = [E]total[S] - [ES][S]

Then collect terms containing [ES] on the left:

[ES] Km + [ES][S] = [E]total[S]

Factor [ES] from the left-hand terms:

[ES](Km + [S]) = [E]total[S]

and finally, divide both sides by (Km + [S]):

[ES] = [E]total [S]/(Km + [S]) (8)

Substitute (8) into (2):

v0 = kcat[E]total [S]/(Km + [S]) (9)

The maximum velocity Vmax occurs when the enzyme is saturated -- that is, when all
enzyme molecules are tied up with S, or [ES] = [E]total. Thus,

Vmax = kcat [E]total (10)

Substitute Vmax into (9) for kcat [E]total:

v0 = Vmax [S]/(Km + [S]) (11)

This equation expresses the initial rate of reaction in terms of a measurable quantity, the
initial substrate concentration. The two kinetic parameters, Vmax and Km , will be different
for every enzyme-substrate pair.

Equation (11), the Michaelis-Menten equation, describes the kinetic behavior of an

enzyme that acts according to the simple model (1). Equation (11) is of the form
y = ax/(b + x) (does this look familiar?)

This is the equation of a rectangular hyperbola, just like the saturation equation for the
binding of oxygen to myoglobin.

Mathematically, the function v0 presents two asymptotes:

* one parallel to the [S] axis at v0 = Vmax , represents the velocity at infinite [S]
(saturation),
* the second parallel to the v0 axis at [S] = - Km, has no physical meaning (no negative
concentrations).

Further analysis reveals the physical meaning of Km: the concentration of substrate at
which the velocity is half Vmax. Indeed, substituting Km for [S] in (11) yields

v0 = 1/2 Vmax.

Thus, a low value for Km may indicate a high affinity of the enzyme for its substrate.

Another physically meaningful limit of this function is found at vanishingly small values
of [S] (--> 0),
where v0 --> Vmax /Km [S].
In this case, the velocity becomes proportional to the (low, relative to Km) substrate
concentration, displaying pseudo-first order kinetics in [S].

The parameter Vmax /Km (or rather its constant part kcat /Km), often referred to as the
catalytic ability of the enzyme, is a direct measure of the efficiency of the enzyme in
transforming the substrate S.

kcat /Km recombines the two traditionally-separated aspects of enzyme catalysis:

* the effectiveness of transformation of bound product (catalysis per se, kcat )

* the effectiveness of productive substrate binding (affinity, 1/Km = k1 /(k-1 + kcat))

Equation (11) means that, for an enzyme acting according to the simple model (1), a plot
of v0 versus [S] will be a rectangular hyperbola. When enzymes exhibit this kinetic
behavior, unless we find other evidence to the contrary, we assume that they act
according to model (1), and call them Michaelis-Menten enzymes.

Relationship between Michaelis-Menten

and Lineweaver-Burk plot.
In this example, we'll make a combination graph commonly used to characterize enzyme activity-
a curve of initial velocity vs. substrate concentration, sometimes referred to as a Michaelis-
Menten plot, with an inset Lineweaver-Burk plot.

Lineweaver-Burk analysis is one method of linearizing substrate-velocity data so as to determine

the kinetic constants Km and Vmax. One creates a secondary, reciprocal plot: 1/velocity vs. 1/
[substrate]. When catalytic activity follows Michaelis-Menten kinetics over the range of substrate
concentrations tested, the Lineweaver-Burk plot is a straight line with

Y intercept = 1/Vmax,
X intercept = -1/Km
slope = Km/Vmax

Now that programs such as Prism easily do nonlinear regression, the best way to determine
Km and Vmax is to fit a hyperbola directly to the substrate-velocity data. Yet the
Lineweaver-Burk plot continues to be a useful visual tool, particularly because of its
characteristic shifts in the presence of various types of inhibitors. So we'll create a
Lineweaver-Burk plot with data points derived from double-reciprocal transformation,
but we'll superimpose a line based upon nonlinear regression analysis, so that it reflects
the best possible estimates of Kd and Bmax.

Range
The most efficient enzymes reach a k2 / KM in the range of 108 - 1010 M−1 s−1. These
enzymes are so efficient they effectively catalyse a reaction each time they encounter a
substrate molecule and have thus reached an upper theoretical limit for efficiency
(diffusion limit); these enzymes have often been termed perfect enzymes.[10]