Michaelis-Menton Kinetics

In a typical enzyme-catalyzed reaction, reactant and product

concentrations are usually hundreds or thousands of times greater than
the enzyme concentration.
Consequently, each enzyme molecule catalyzes the conversion to
product of many reactant molecules during a period .
.
The catalytic event that converts substrate (in biochemical reactions,
reactants are commonly known as substrates) to product involves the
formation of a transition state, and it occurs most easily at a specific
binding site on the enzyme.
This site, called the catalytic site of the enzyme.
It has been evolutionarily structured to provide specific, high-affinity binding of
substrate(s) and to provide an environment that favors the catalytic events.
The complex that forms when substrate(s) and enzyme combine is
called the enzyme substrate (ES) complex.
Reaction products arise when the ES complex breaks down releasing
free enzyme.
Between the binding of substrate to enzyme, and the reappearance of
free enzyme and product, a series of complex events take place.
At a minimum; an ES complex must be formed; this complex must pass
to the transition state (ES*); and the transition state complex must
advance to an enzyme product complex (EP).

The latter is finally competent to dissociate to product and free enzyme.

The series of events can be shown as:
E + S <---> ES <---> ES* <---> EP <---> E + P
The kinetics of simple reactions like that above were first characterized
by biochemists Michaelis and Menten.
The concepts underlying their analysis of enzyme kinetics continue to
provide the cornerstone for understanding metabolism today.

Except for initial build-up of [ES] at the beginning of the reaction (a few
milliseconds) the [ES] remains approximately constant until substrate is
nearly all used up.

So, although we can solve the above expression for [ES] as a function of
[E] and [S], even though we can monitor [S] at time "t", it is usually
much more difficult to measure [E] at time "t".
Therefore, the more directly measurable total enzyme concentration [E]T
- which is the initial amount of enzyme introduced to the system is
introduced.
From conservation of mass (at constant volume), it is easy to see that:
So, if we substitute
[E] = [E]T - [ES]
into our steady-state expression mentioned,
k1 (E)(S)= k-1(ES)+k2(ES)
the result is:
k1 [(Et)-(ES)](S)=k-1(ES)+k2(ES)
Rearranging the equation we obtain:
(S) [(ET)-(ES)]/(ES) = (k-1 + k2)/k1
where
(k-1+k2)/k1 = Km

Solving for (ES) we obtain:

Multiplying both sides with k2 we will have

Thus, the above expression is commonly written in terms of the "initial

velocity" (v0) as:
Vo = k2 (ES)
As the [S] becomes very large, the velocity of reaction will not
increase indefinitely, but - for a fixed amount of [E]T - will reach a
limiting value termed Vmax the maximal velocity.
Vmax = k2 (ET)

Substituting Vmax and Vo in our boxed expression above gives the "usual"
form - termed the Michaelis-Menten equation:

This expression is the basic equation of enzyme kinetics.

The Michaelis-Menten equation: is a quantitative description of the
relationship among the rate of an enzyme- catalyzed reaction [v o] , the
concentration of substrate [S] and two constants, Vmax and Km (which are
set by the particular equation).
The Michaelis-Menten equation can be used to demonstrate that at the
substrate concentration that produces exactly half of the maximum
reaction rate, i.e.,1/2 Vmax, the substrate concentration is numerically
equal to Km.

Rearranging the Michaelis-Menton equation leads to:

From this equation it should be apparent that when the substrate

concentration is half that required to support the maximum rate of
reaction, the observed rate, vo, will, be equal to Vmax divided by 2; in
other words,
vo = [Vmax/2].
At this substrate concentration Vmax/Vo will be exactly equal to 2,
with the result that
[S] = Km

To avoid dealing with curvilinear plots of enzyme catalyzed reactions,

biochemists Lineweaver and Burk introduced an analysis of enzyme
kinetics based on the following rearrangement of the Michaelis-Menten
equation:

Plots of 1/v versus 1/[S] yield straight lines having a slope of Km/Vmax
and an intercept on the ordinate at 1/Vmax.

An alternative linear transformation of the Michaelis-Menten equation is

the Eadie-Hofstee transformation:
v/[S] = -v [1/Km] + [Vmax/Km]
and when v/[S] is plotted on the y-axis versus v on the x-axis, the
result is a linear plot with a slope of -1/Km and the value Vmax/Km as
the intercept on the y-axis and Vmax as the intercept on the x-axis.

This fact provides a simple yet powerful bioanalytical tool that has been
used to characterize both normal and altered enzymes, such as those
that produce the symptoms of genetic diseases.
In order to determine the amount of an enzyme present in a sample of
tissue, it is obviously essential to ensure that the limiting factor is the activity of
the enzyme itself, and not the amount of substrate available.
This means that the concentration of substrate must be high enough to ensure
that the enzyme is acting at Vmax.
In practice, it is usual to use a concentration of substrate about 10 - 20-fold
higher than the Km in order to determine the activity of an enzyme in a sample.