BS Chemistry Semester 6
Biochemistry III
Submitted To:
Mam Mariyam
Submitted By:
Hifza Habib
Roll No: 28
Department Of Chemistry
University of Central Punjab
Table of content:
Sr. Content Pages
1 History 2
2 Introduction 2
3 General principles 3
4 Enzyme Assays 4
5 Single substrate reaction 5
6 Subtrate complex 5
7 The velocity equation 6
8 Lineweaver Burk plot 7
9 Derivation 8
10 Enzyme kinetics Mechanism 9
11 References 13
History:
In 1902 Victor Henri proposed a quantitative theory of enzyme
kinetics, but at the time the experimental significance of
the hydrogen ion concentration was not yet recognized.
After Peter Lauritz Sørensen had defined the logarithmic pHscale and
introduced the concept of buffering in 1909 the
German chemist Leonor Michaelis and his Canadian
postdoc Maud Leonora Menten repeated Henri's experiments
and confirmed his equation, which is now generally referred to
as Michaelis-Menten kinetics.
Their work was further developed by G. E. Briggs and J. B. S.
Haldane, who derived kinetic equations that are still widely
considered today a starting point in modeling enzymatic activity.
Introduction:
Enzyme kinetics is the study of the chemical reactions that
are catalysed by enzymes.
In enzyme kinetics, the reaction rate is measured and the
effects of varying the conditions of the reaction are
investigated.
Enzymes are usually protein molecules that manipulate other
molecules—the enzymes' substrates.
These target molecules bind to an enzyme's active site and are
transformed into products through a series of steps known as
the enzymatic mechanism.
E + S ES ES* EP E + P
General Principles:
The reaction catalysed by an enzyme uses exactly the same reactants and
produces exactly the same products as the uncatalysed reaction.
Like other catalysts, enzymes do not alter the position of equilibrium between
substrates and products.
However, unlike uncatalysed chemical reactions, enzyme-catalysed reactions
display saturation kinetics.
For a given enzyme concentration and for relatively low substrate
concentrations, the reaction rate increases linearly with substrate
concentration; the enzyme molecules are largely free to catalyse the reaction,
and increasing substrate concentration means an increasing rate at which the
enzyme and substrate molecules encounter one another.
However, at relatively high substrate concentrations, the reaction
rate asymptotically approaches the theoretical maximum; the enzyme active
sites are almost all occupied and the reaction rate is determined by the intrinsic
turnover rate of the enzyme.
Enzyme Assays:
Enzyme assays are laboratory procedures that measure the rate of enzyme
reactions.
Since enzymes are not consumed by the reactions they catalyse, enzyme
assays usually follow changes in the concentration of either substrates or
products to measure the rate of reaction.
There are many methods of measurement.
Spectrophotometric assays observe change in the absorbance of light
between products and reactants; radiometric assays involve the incorporation
or release of radioactivity to measure the amount of product made over time.
Spectrophotometric assays are most convenient since they allow the rate of
the reaction to be measured continuously.
Although radiometric assays require the removal and counting of samples
(i.e., they are discontinuous assays) they are usually extremely sensitive and
can measure very low levels of enzyme activity.
Single-Substrate Reactions:
Michaelis–Menten kinetics:
As enzyme-catalysed reactions are saturable, their rate of
catalysis does not show a linear response to increasing
substrate.
If the initial rate of the reaction is measured over a range of
substrate concentrations (denoted as [S]), the initial reaction
rate increases as [S] increases, as shown on the right.
However, as [S] gets higher, the enzyme becomes saturated
with substrate and the initial rate reaches Vmax, the enzyme's
maximum rate.
There is an initial bimolecular reaction between the
enzyme E and substrate S to form the enzyme–substrate
complex ES.
The rate of enzymatic reaction increases with the increase
of the substrate concentration up to a certain level called
Vmax; at Vmax, increase in substrate concentration does not
cause any increase in reaction rate as there no more
enzyme (E) available for reacting with substrate (S).
Here, the rate of reaction becomes dependent on the ES complex and the
reaction becomes a unimolecular reaction with an order of zero
Enzyme Kinetics as an Approach to
Understanding Mechanism:
Biochemists commonly use several approaches to study the
mechanism of action of purified enzymes.
The three dimensional structure of the protein provides important
information, which is enhanced by classical protein chemistry and
modern methods of site-directed mutagenesis.
These technologies permit enzymologists to examine the role of
individual amino acids in enzyme structure and action.
However, the oldest approach to understanding enzyme mechanisms,
and the one that remains most important, is to determine the rate of a
reaction and how it changes in response to changes in experimental
parameters, a discipline known as enzyme kinetics.
References:
Biochemistry – Lehninger ( Nelson and Cox)
Sixth Edition
