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ENZYME KINETICS

Enzyme kinetics is the study of the rates of chemical reactions that are catalysed
by enzymes. The study of an enzyme's kinetics provides insights into the catalytic
mechanism of this enzyme, its role in metabolism, how its activity is controlled in
the cell and how drugs and poisons can inhibit its activity. (General Chemistry, 2007)

Enzyme kinetics is the study of the binding affinities of substrates and inhibitors and
the maximal catalytic rates that can be achieved. (Engelking)

An essential feature of enzyme-catalyzed reactions is saturation: at increasing


concentrations of substrates the rate increases and approaches a limit where there
is no dependence of rate on concentration

Michaelis-Menten Kinetics

Two 20th century scientists, Leonor Michaelis and Maud Leonora Menten, proposed
the model known as Michaelis-Menten Kinetics to account for enzymatic dynamics.
The model serves to explain how an enzyme can cause kinetic rate enhancement of
a reaction and explains how reaction rates depends on the concentration of enzyme
and substrate. (Michaelis-Menten Kinetics)

The general reaction scheme of an enzyme-catalyzed reaction is as follows:

The Michaelis-Menten equation arises from the general equation for an enzymatic
reaction: E + S ↔ ES ↔ E + P, where E is the enzyme, S is the substrate, ES is the
enzyme-substrate complex, and P is the product. Thus, the enzyme combines with
the substrate in order to form the ES complex, which in turn converts to product
while preserving the enzyme.
Table 1: Model parameters

Rate Reaction
Constant

k1 The binding of the enzyme to the substrate forming the enzyme substrate
complex.

k2 Catalytic rate; the catalysis reaction producing the final reaction product
and regenerating the free enzyme. This is the rate limiting step.

k-1 The dissociation of the enzyme-substrate complex to free enzyme and


substrate .

k-2 The reverse reaction of catalysis.

Therefore, the ES complex may dissolve back into the enzyme and substrate, or
move forward to form product.

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

The M-M equation was derived in part by making several assumptions. An important
one was: the concentration of substrate must be much greater than the
enzyme concentration.
In the situation where [S] >> [E] and at initial velocity rates, it is assumed that the
changes in the concentration of the intermediate ES complex are very small over
time (vo). This condition is termed a steady-state rate, and is referred to as steady-
state kinetics. Therefore, it follows that the rate of ES formation will be equal
to the rate ES breakdown.

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.

Km implies that half of the active sites on the enzymes are filled. Different enzymes
have different Km values. They typically range from 10-1 to 10-7 M. The factors that
affect Km are:
 pH
 temperature
 ionic strengths
 the nature of the substrate

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.This small Km will approach
Vmax more quickly than high Km value.

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 also known as the turnover number as it represents the maximum number of
substrate molecules that the enzyme can 'turn over' to product in a set time (e.g. the
turnover numbers of a-amylase, glucoamylase and glucose isomerase are 500 s-1,
160 s-1 and 3 s-1respectively; an enzyme with a relative molecular mass of 60000
and specific activity 1 U mg-1 has a turnover number of 1 s-1). 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.

The ratio kcat/Km determines the relative rate of reaction at low substrate
concentrations, and is known as the specificity constant.

When Kcat/ Km, it gives us a measure of enzyme efficiency with a unit of


1/(Molarity*second)= L/ (mol*s). The enzyme efficiency can be increased as Kcat
has high turnover and a small number of Km.
Then, at

Therefore, Km is equal to the concentration of the substrate when the rate is half of
the maximum velocity. From the Michaelis Menten Kinetic equation, we have many
different ways to find Km and Vmax such as the Lineweaver-Burk plot, Hanes-Woolf
plot, and Eadie-Hofstee plot, etc.

In a mathematical description of enzyme action developed by Leonor Michaelis and


Maud Menten in 1913, two constants, Vmax and Km, play an important role. These
constants are important to know, both to understand enzyme activity on the
macroscale and to understand the effects of different types of enzyme inhibitors.

The table shows the Km and Vmax of some common enzymes:

Enzyme Km Vmax

Carbonic Anhydrase 8000 600,000

Chymotrypsin 5000 100

Penicillinase 50 2000

Lysozyme 6 0.5

Taking the reciprocal of both side of the Michaelis-Menten equation


gives:
To determined the values of KM and Vmax. The double-reciprocal of Michaels-
Menten equation could be used.

Lineweaver-Burk Plot

The Lineweaver-Burk plot or double reciprocal plot is common way of illustrating


kinetic data. This is produced by taking the reciprocal of both sides of the Michaelis–
Menten equation. As shown on the right, this is a linear form of the Michaelis–
Menten equation and produces a straight line with the equation y = mx + b with a y-
intercept equivalent to 1/Vmax and an x-intercept of the graph representing -1/Km.

Reaction Order Note

When [S]<<Km,

This means that the rate and the substrate concentration are directly proportional to
each other. The reaction is first-order kinetics.
When [S]>>Km,
This means that the rate is equal to the maximum velocity and is independent of the
substrate concentration. The reaction is zero-order kinetics.

Enzyme Inhibition

Enzyme inhibitors are molecules that reduce or abolish enzyme activity. These are
either reversible (i.e., removal of the inhibitor restores enzyme activity)
or irreversible (i.e., the inhibitor permanently inactivates the enzyme).

1. REVERSIBLE INHIBITORS

Reversible inhibitors interact with enzyme via non covalent associations.

THREE REVERSIBLE INHIBITORS:

 Competitive

Competitive inhibitors are molecules that look like substrates and they
bind to active site and slow down the reactions. Therefore, competitive inhibitors
increase Km value (decrease affinity, less chance the substrates can go to active
site), and Vmax stays the same. On double reciprocal plot, competitive inhibitor
shifts the x-axis (1/[s]) to the right towards zero compared to the slope with no
inhibitor present.

 Uncompetitive

Uncompetitive inhibitors can bind close to the active site but don't
occupy the active site. As a result, uncompetitive inhibitors lower Km
(increase affinity) and lower Vmax. On double reciprocal plot, x-axis (1/[s]) is
shifted to the left and up on the y-axis (1/V) compared to the slope with no
inhibitor.

 Non-competitive.
Non-competitive inhibitors are not bind to the active site but
somewhere on that enzyme which changes its activity. It has the same Km
but lower Vmax to those with no inhibitors. On the double reciprocal plot, the
slope goes higher on y-axis (1/V) than the one with no inhibitor. Km value is
numerically equal to the substrate concentration at which the half of the
enzyme molecules are associated with substrate. km value is an index of the
affinity of enzyme for its particular substrate.

2. IRREVERSIBLE INHIBITORS
Enzyme inhibitors can also irreversibly inactivate enzymes, usually by covalent
associations. These reactions follow exponential decay functions and are usually
saturable. Below saturation, they follow first order kinetics with respect to inhibitor.

Temperature Effects

As shown in Figure 13, the reaction rate increases with temperature to a maximum
level, then abruptly declines with further increase of temperature. Because most
animal enzymes rapidly become denatured at temperatures above 40°C, most
enzyme determinations are carried out somewhat below that temperature.

Over a period of time, enzymes will be deactivated at even moderate temperatures.


Storage of enzymes at 5°C or below is generally the most suitable. Some enzymes
lose their activity when frozen.

Effects of pH
Enzymes are affected by changes in pH. The most favorable pH value - the point
where the enzyme is most active - is known as the optimum pH. This is graphically
illustrated in Figure 14.
Extremely high or low pH values generally result in complete loss of activity for most
enzymes. pH is also a factor in the stability of enzymes. As with activity, for each
enzyme there is also a region of pH optimal stability.
The optimum pH value will vary greatly from one enzyme to another, as Table II
shows:

Table II: pH for Optimum Activity

Enzyme pH Optimum

Lipase (pancreas) 8.0

Lipase (stomach) 4.0 - 5.0

Lipase (castor oil) 4.7

Pepsin 1.5 - 1.6

Trypsin 7.8 - 8.7

Urease 7.0

Invertase 4.5

Maltase 6.1 - 6.8

Amylase (pancreas) 6.7 - 7.0

Amylase (malt) 4.6 - 5.2

Catalase 7.0
References:
Biomolecules:. (n.d.). Retrieved from www.chem.wisc.edu:
https://www.chem.wisc.edu/deptfiles/genchem/netorial/modules/biomolecules/modul
es/enzymes/enzyme4.htm

Enzyme Kinetics. In L. R. Engelking, Textbook of Veterinary Physiological Chemistry


(Third Edition), 2015. Elsevier's Integrated Review Pharmacology (Second Edition).

Enzyme Kinetics. (n.d.). Retrieved 2012, from


https://www.sciencedirect.com/topics/neuroscience/enzyme-kinetics

General Chemistry. (2007). Retrieved from Enzymes Kinetics:


https://www.cs.mcgill.ca/~rwest/wikispeedia/wpcd/wp/e/Enzyme_kinetics.htm

Introduction to Enzymes. (n.d.). Retrieved 2019, from Worthington Biochemical


Corporation cartPlace Order: The optimum pH value will vary greatly from one
enzyme to another, as Table II shows:

Michaelis-Menten Kinetics. (n.d.). Retrieved April 24, 2019, from chem.libretexts.org:


https://chem.libretexts.org/Bookshelves/Biological_Chemistry/Supplemental_Module
s_(Biological_Chemistry)/Enzymes/Enzymatic_Kinetics/Michaelis-Menten_Kinetics

Structural Biochemistry/Enzyme/Michaelis and Menten Equation. (n.d.). Retrieved


from Wikibooks:
https://en.m.wikibooks.org/wiki/Structural_Biochemistry/Enzyme/Michaelis_and_Men
ten_Equation?fbclid=IwAR06IvAHn8TaeHgQwLaXDG0diHYcO1B1bEJbPLIKImsICM
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