Enzymes Kinetic

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 Enzyme kinetics is the study of the chemical reactions that are catalyzed by enzymes
In enzyme kinetics, the reaction rate is measured and the effects of varying the
conditions of the reaction are investigated. Studying an enzyme's kinetics in this way
can reveal the catalytic mechanism of the enzyme, its role in metabolism, how its
activity is controlled, and how a drug or an agonist might inhibit the enzyme.
Rate Limiting Steps
The rate limiting step in any reaction is its slowest step. In enzymatic reactions, the
conversion of the enzyme-substrate complex to the product is normally rate
limiting. The rate of this step (and therefore the entire enzymatic reaction) is
directly proportional to the concentration of ES
The concentration of ES changes as the reaction progresses
Therefore, the rate of product formation also changes over time. When the reaction
reaches equilibrium (steady state) the concentration of ES (and therefore the rate)
remains relatively constant.
Reaction Kinetics
When an enzyme is added to a lot of substrate, the reaction that follows occurs in
three stages with distinct kinetics:
Pre-steady state
Steady state (equilibrium)
Post-steady

The pre-steady state phase is very short as equilibrium is reached within

microseconds.
If you measure the rate in the first few seconds of-aMenton
Michaelis reaction (V ) you are actually
Kinetics
0
measuring the steady state.
Two terms that are important within Michaelis-Menton Kinetics are:
This is–the
Vmax rate
the used in Michaelis-Menton
maximum Kinetics.
rate of reaction when all enzyme active sites are saturated
substrate
Km – the substrate concentration that gives half maximal velocity. •
Km is a measure of the affinity an enzyme has for its substrate, as a lower Km me
that less of the substrate is required to reach half of Vmax.

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 ‫مهم‬
Michaelis-Menton Kinetics
Two terms that are important within Michaelis-Menton Kinetics
are:
max – the maximum rate of reaction when all enzyme active
sites are saturated with substrate
Km – the substrate concentration that gives half maximal •
velocity.
Km is a measure of the affinity an enzyme has for its substrate,
as a lower Km means that less of the substrate is required to reach
half of Vmax .

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‫فقط ا‪+‬عادلة مطلوبة‬

‫مهم‬
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This equation concerns the steady state of an enzymatic reaction with one
substrate, and is given by:

It describes how the initial rate of reaction, V0, is affected by the initial
substrate concentration, [S]0
It only looks at the start of the reaction.

This allows it to ignore the reverse reaction where substrate is formed from
product. This is because at the start of the reaction there is no product present
to become substrate.

The plot of rate against concentration has the shape of a rectangular

hyperbola. However, a more useful representation of Michaelis–Menten
kinetics is a graph called a Lineweaver–Burk plot.

The equation used to generate this plot is given by

This allows an easier interpretation of various quantities from the graph, such
as the presence of an inhibitor.
The Michaelis-Menten equation arises from the general equation for an
enzymatic reaction: E + S ↔ ES ↔ E + P, Thus, the enzyme combines with
the substrate in order to form the ES complex, which in turn converts to
product while preserving the enzyme
The rate of the forward reaction from E + S to ES may be termed k1, and the
reverse reaction as k-1.
Likewise, for the reaction from the ES complex to E and P, the forward
reaction rate is k2, and the reverse is k-2. Therefore, the ES complex may
dissolve back into the enzyme and substrate, or move forward to form
product.

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At initial reaction time, when t ≈ 0, little product formation occurs, therefore the
backward reaction rate of k-2 may be neglected. The new reaction becomes:

E + S ↔ ES → E + P

Assuming steady state, the following rate equations may be written

as
Rate of formation of ES = k1[E][S]
Rate of breakdown of ES = (k-1 + k2) [ES]
and set equal to each other (Note that the brackets represent
concentrations). Therefore
k1[E][S] = (k-1 + k2) [ES]
Rearranging terms,
[E][S]/[ES] = (k-1 + k2)/k1

The fraction [E][S]/[ES] has been coined Km, or the Michaelis constant

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According to Michaelis-Menten's kinetics equations, at low concentrations of
substrate, [S], the concentration is almost negligible in the denominator as KM >>
[S], so the equation is essentially
V0 = Vmax [S]/KM
which resembles a first order reaction
At High substrate concentrations, [S] >> KM, and thus the term [S]/([S] + KM)
becomes essentially one and the initial velocity approached Vmax, which resembles
zero order reaction .
The Michaelis-Menten equation is In this equation
V0 is the initial velocity of the reaction
Vmax is the maximal rate of the reaction
[Substrate] is the concentration of the substrate

Km is the Michaelis-Menten constant which shows the concentration of

the substrate when the reaction velocity is equal to one half of the
maximal velocity for the reaction

It can also be thought of as a measure of how well a substrate complexes

with a given enzyme, otherwise known as its binding affinity
An equation with a low Km value indicates a large binding affinity, as the
reaction will approach Vmax more rapidly. An equation with a high Km
indicates that the enzyme does not bind as efficiently with the substrate,
and Vmax will only be reached if the substrate concentration is high
enough to saturate the enzyme.

As the concentration of substrates increases at constant enzyme

concentration, the active sites on the protein will be occupied as the
reaction is proceeding.

When all the active sites have been occupied, the reaction is complete,
which means that the enzyme is at its maximum capacity and increasing
the concentration of substrate will not increase the rate of turnover.

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Vmax is equal to the product of the catalyst rate constant (kcat) and the
concentration of the enzyme.

The Michaelis-Menten equation can then be rewritten as V= Kcat

[Enzyme] [S] / (Km + [S])
Kcat is equal to K2, and it measures the number of substrate molecules
"turned over" by enzyme per second.

The unit of Kcat is in 1/sec. The reciprocal of Kcat is then the time
required by an enzyme to "turn over" a substrate molecule. The higher
the Kcat is, the more substrates get turned over in one second.
Km is the concentration of substrates when the reaction reaches half of
Vmax.
A small Km indicates high affinity since it means the reaction can reach
half of Vmax in a small number of substrate concentration. This small
Km will approach Vmax more quickly than high Km value
The turnover number of an enzyme, is the number of substrate molecules
converted into product by an enzyme molecule in a unit time when the
enzyme is fully saturated with substrate.

In enzyme kinetics, we are interested to know how many maximum

molecules of substrate can be converted into product per catalytic site of
a given concentration of enzyme per unit time. kcat=Vmax / Et(1)
with kcat = Turnover number, Vmax= Maximum rate of reaction when
all the enzyme catalytic sites are saturated with substrate and
ET =Total enzyme concentration or concentration of total enzyme
catalytic sites.
The units of Turn over number (kcat) are kcat
= (moles of product/sec)/ (moles of enzyme) or sec-1.
Leonor Michaelis and Maud Menten introduced a mathematical
illustration to describe the action of enzymes with two constants, Vmax
and Km.
The maximal velocity (Vmax) refers to the point at which the increase the
concentration of the substrate does not increase the rate of a reaction
catalyzed by an enzyme. This occurs because the substrate molecules
saturate the active sites of the enzyme and are not able to form more
complexes with the enzyme. This value is given as a rate (mmol/s), which
is the maximum velocity of the reaction when the enzyme is saturated.

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Since, Vmax is achieved at infinite substrate concentration, it is
impossible to estimate Vmax and hence Km from a hyperbolic plot
Because of this difficulty, the Michaelis–Menten equation was
transformed into an equation for a straight line by Lineweaver and
Burk.

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Where V is the reaction velocity (the reaction rate), Km is
the Michaelis–Menten constant, Vmax is the maximum reaction velocity, and [S] is
the substrate concentration.

It gives a straight line, with the intercept on the y-axis equal to

1/Vmax, and the intercept on the x-axis equal to Km/Vmax. The
slope of the line is equal to Km/Vmax
Vmax and Km can be determined experimentally by
measuring V0 at different substrate concentrations. Then a
double reciprocal or Lineweaver–Burk plot of 1/V0 against 1/[S]
is made.
Lineweaver-Burk) plot is created by plotting the inverse initial
velocity (1/V0) as a function of the inverse of the substrate
concentration (1/[S]). ... This plot is a useful way to determined
different inhibitors such as competitive, uncompetitive, and
noncompetitive.

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The Michaelis constant (Km) is the concentration of the substrate
when half of the active binding sites of an enzyme are occupied
by the substrate.
The constant helps to depict the affinity of the enzyme for their
substrate
This value is given as the concentration of the substrate (mM) at
half of Vmax
An enzyme with a high Km has a low affinity for the substrate,
and a high concentration of the substrate is needed in order for
the enzyme to become saturated. Conversely, an enzyme with a
high Km has a high affinity for the substrate and the enzyme may
become saturated even with a low amount of substrate.

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Allosteric enzymes do not obey Michaelis-Menten kinetics. ... Thus,
allosteric enzymes show the sigmodial curve. The plot for reaction
velocity, vo, versus the substrate concentration does not exhibit the
hyperbolic plot predicted using the Michaelis-Menten equation.

An allosteric enzyme is an enzyme that contains a region to which small,

regulatory molecules ("effectors") may bind in addition to and separate
from the substrate binding site and thereby affect the catalytic activity.
On binding the effector, the catalytic activity of the enzyme towards the
substrate may be enhanced, in which case the effector is an activator, or
reduced, in which case it is a de-activator or inhibitor.

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 Allosteric Regulation
Allosteric regulations the regulation of activities of an enzyme or a
protein caused by the binding of modulators ( or effectors) at the site
other than the active site of the enzyme or protein. There are two effects
of Allosteric regulations and two effectors are may be available for the
effects
Effectors maybe-feed-forward activators or feedback inhibitors.
Allosteric--derived from the Greek--meaning other shape • Characterized
by sigmoidal kinetics (as opposed to Michaelis-Menton kinetics) – show
cooperativity – quaternary structure – (note: hemoglobin shows allosteric
behavior although it is not an enzyme) •
Conformational change usually involves binding small effector molecules
that are not substrates- -these bind to an allosteric site on the enzyme that
result in rapid changes in enzyme activity

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An enzyme inhibitor is a molecule that binds to an enzyme and decreases its activity
By binding to enzymes' active sites, inhibitors reduce the compatibility of substrate
and enzyme and this leads to the inhibition of Enzyme-Substrate complexes'
formation, preventing the catalyzation of reactions and decreasing (at times to zero)
the amount of product produced by a reaction
It can be said that as the concentration of enzyme inhibitors increases, the rate of
enzyme activity decreases, and thus, the amount of product produced is inversely
proportional to the concentration of inhibitor molecules

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 Reversible inhibitors
Reversible inhibitors attach to enzymes with non-covalent interactions such as
hydrogen bonds, hydrophobic interactions and ionic bonds. Multiple weak bonds
between the inhibitor and the active site combine to produce strong and specific
binding. In contrast to substrates and irreversible inhibitors, reversible inhibitors
generally do not undergo chemical reactions when bound to the enzyme and can be
easily removed by dilution or dialysis .
There are four kinds of reversible enzyme inhibitors. They are classified according
to the effect of varying the concentration of the enzyme's substrate on the inhibitor
In competitive inhibition, the substrate and inhibitor cannot bind to the enzyme
at the same time.(Competitive inhibitors are often similar in structure to the real
substrate )
This usually results from the inhibitor having an affinity for the active site of an
enzyme where the substrate also binds; the substrate and inhibitor compete for
access to the enzyme's active site. This type of inhibition can be overcome by
sufficiently high concentrations of substrate (Vmax remains constant), i.e., by out-
competing the inhibitor.
However, the apparent Km will increase as it takes a higher concentration of the
substrate to reach the Km point, or half the Vmax.

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Line-Weaver Burk Plot of competitive inhibition

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In uncompetitive inhibition
The inhibitor binds only to the substrate-enzyme complex
This type of inhibition causes Vmax to decrease (maximum velocity decreases as a
result of removing activated complex) and Km to decrease (due to better binding
efficiency as a result of Le Chatelier's principle and the effective elimination of the
ES complex thus decreasing the Km which indicates a higher binding affinity).

In non-competitive inhibition, the binding of the inhibitor to the enzyme reduces its
activity but does not affect the binding of substrate. As a result, the extent of
inhibition depends only on the concentration of the inhibitor. Vmax will decrease
due to the inability for the reaction to proceed as efficiently, but Km will remain the
same as the actual binding of the substrate, by definition, will still function properly.
Line-Weaver Burk Plot of noncompetitive inhibition.

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In mixed inhibition, the inhibitor can bind to the enzyme at the same
time as the enzyme's substrate. However, the binding of the inhibitor
affects the binding of the substrate, and vice versa. This type of
inhibition can be reduced, but not overcome by increasing
concentrations of substrate
Although it is possible for mixed-type inhibitors to bind in the active
site, this type of inhibition generally results from an allosteric effect
where the inhibitor binds to a different site on an enzyme. Inhibitor
binding to this allosteric site changes the conformation (i.e., tertiary
structure or three-dimensional shape) of the enzyme so that the
affinity of the substrate for the active site is reduced.

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