enzyme kinetics
TRANSCRIPT
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Fundamentals of
*Enzymes = biocatalysts.
*Kinetics = study of motion and its causes.
*Enzyme + kinetic = The study of biochemical reaction
rates catalyzed by an enzyme.
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m+n = order of reaction
Catalysts lower the activation energy of the
reaction.
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Enzyme Kinetics
*The primary function of enzymes is to enhance rates of
reactions so that they are compatible with the needs of the
organism.
*To understand how enzymes function, we need a kinetic
description of their activity.
*For many enzymes, the rate of catalysis V0, which is defined as
the number of moles of product formed per second, varies with
the substrate concentration [S]
*Different theories and plots have been proposed by various
scientists some of which are Michaelis –Menton plot, Line
weaver plot , Hill’s plot and so on.
*It shows that the maximal velocity (Vmax) is approached asymptotically.
*(KM) is the substrate concentration yielding a velocity of Vmax/2
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Significance of Km
KM is the concentration of substrate at which half the active
sites are filled. It provides a measure of the substrate
concentration required for significant catalysis to occur.
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Consider the following equation :
**Consider a case in which k-1 >>> k2
Under such circumstances, the ES --- E + S much more rapidly than product is
formed.
So,
**When this condition is met, KM is a measure of the strength of the ES complex:
a high KM indicates weak binding; a low KM indicates strong binding.
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*The maximal rate, Vmax, reveals the turnover number of
an enzyme
* It 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.
*The maximal rate, Vmax, reveals the turnover number of
an enzyme if the concentration of active sites [E]T is
known,.
*The enzyme efficiency can be increased as Kcat has high turnover
and a small number of Km.
Line Weaver Burk plot
In order to determine Km and Vmax, the Michaelis
Menten equation is rewritten as :
•The y-intercept is equivalent to the
•inverse of Vmax.
•The x-intercept of the graph
represents −1/Km.
Significance of the plots :
.1)For determining inhibition
*For competitive inhibitors, the KM will increase without changing
the Vmax value.
*This means that the two graphs will have the same y-intercept
as shown below. *However the new x-intercept may be quite
elusive.
www.nbs.csudh.edu
Significance of the plots :
.1)For determining inhibition
*For competitive inhibitors, the KM will increase without changing
the Vmax value.
*This means that the two graphs will have the same y-intercept
as shown below. *However the new x-intercept may be quite
elusive.
www.nbs.csudh.edu
Determining uncompetitive
inhibition :
An uncompetitive inhibitor binds to the enzyme and
enhances the binding of substrate (so reducing Km),
but the resultant enzyme-inhibitor-substrate complex
only undergoes reaction to form the product slowly,
so that Vmax is also reduced:
Determining uncompetitive
inhibition :
An uncompetitive inhibitor binds to the enzyme and
enhances the binding of substrate (so reducing Km),
but the resultant enzyme-inhibitor-substrate complex
only undergoes reaction to form the product slowly,
so that Vmax is also reduced:
www.ucl.ac.uk
Determining uncompetitive
inhibition :
An uncompetitive inhibitor binds to the enzyme and
enhances the binding of substrate (so reducing Km),
but the resultant enzyme-inhibitor-substrate complex
only undergoes reaction to form the product slowly,
so that Vmax is also reduced:
Increased
1/Km
Increase
d
1/Vmax
www.ucl.ac.uk
For noncompetitive inhibitors, the inhibitor can bind to the enzyme before the substrate can bind to the binding site.
It does not wait for the ES complex to form.
The inhibition will cause a decrease in Vmax value while the KM is unaffected.
Determining non competitive inhibition :
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Applications of Enzyme Kinetic’s study
One of the applications of enzyme kinetics is the
determination of dissociation constants for antigen-antibody
interactions in solution.
Applications of Enzyme Kinetic’s study
One of the applications of enzyme kinetics is the
determination of dissociation constants for antigen-antibody
interactions in solution.
According to researchers double reciprocal plots of Elisa
signals versus antigen concentration helps in studying antigen
antibody binding and hence aids drug designing.(1)
Applications of Enzyme Kinetic’s study
One of the applications of enzyme kinetics is the
determination of dissociation constants for antigen-antibody
interactions in solution.
According to researchers double reciprocal plots of Elisa
signals versus antigen concentration helps in studying antigen
antibody binding and hence aids drug designing.(1)
Kinetics study also helps in studying drug metabolism
*Since the enzyme’s activity is affected by concentration, ph etc
studying the kinetics might help in providing suitable conditions for
drug to be efficient.
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References :
sst-web.tees.ac.uk
(3)sst-web.tees.ac.uk
(2)www.pearsonhighered.com
(4) www1.lsbu.ac.uk/
(7)Ewiki book : Structural
Biochemistry/Enzyme/Double-Reciprocal
plot: Lineweaver Burk plote
(6)www.ncbi.nlm.nih.gov/
(9)www.ucl.ac.uk
(5) csrri.iit.edu/
(8)chemwiki.ucdavis.edu/
(1)The application of enzyme kinetics to the determination of dissociation constants for
antigen-antibody interactions in solution
Marc F. Hoylaerts, Alex Bollen, Marc E. De Broe
