enzyme kinetics

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2014-15School of biosciences

Bioenergetics and biophysicsBSBT 213

Presented by :Sakshi SaxenaASU2013010200124B.Tech (Biotechnology)IIIrd sem

ccl.northwestern.edu Fundamentals of Introduction*Enzymes = biocatalysts.

*Kinetics = study of motion and its causes.

*Enzyme + kinetic = The study of biochemical reaction rates catalyzed by an enzyme.

beerandwinejournal.comwww.itqb.unl.pt

Kinetics basicsm+n = order of reactionWhat catalysts do?Catalysts lower the activation energy of the reaction. schoolworkhelper.net

SESPEa = minimum energy that is required for the conversion of reactants into product.5 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 catalysisV0, 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 , Hills plot and so on.Asymtote : distance between line and curve approaches 06

*It shows that the maximal velocity (Vmax) is approached asymptotically.*(KM) is the substrate concentration yielding a velocity ofVmax/2plantphys.infoSignificance of Km KMis the concentration of substrate at which half the active sites are filled. Itprovides a measure of the substrate concentration required for significant catalysis to occur.

www.chem4kids.comKMis related to the rate constants of the individual steps in the catalytic scheme8 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,KMis a measure of the strength of the ES complex: a highKMindicates weak binding; a lowKM indicates strong binding.www.ncbi.nlm.nih.gov*The maximal rate,Vmax, reveals theturnover numberof an enzyme

* It isthe 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]Tis known,.

Significance of Vmax*The enzyme efficiency can be increased as Kcat has high turnover and a small number of Km.Line Weaver Burk plotIn order to determine Km and Vmax, the Michaelis Menten equation is rewritten as :

They-interceptis equivalent to the inverse ofVmax.

The x-interceptof the graph represents 1/Km.Significance of the plots :.1)For determining inhibition *For competitive inhibitors, the KMwill increase without changing the Vmaxvalue. *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 KMwill increase without changing the Vmaxvalue. *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

The antibiotic sulfanilamide is similar in structure to para-aminobenzoic acid (PABA), an intermediate in the biosynthetic pathway for folic acid. Sulfanilamide can competitively inhibit the enzyme that has PABA as it's normal substrate by competitively occupying the active site of the enzyme.13Determining 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.ukLithium and the phosphoinositide cycle15Determining 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/KmIncreased 1/Vmaxwww.ucl.ac.ukFor 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 Vmaxvalue while the KMis unaffected.Determining non competitive inhibition :

chelseaharripersad.wordpress.comNoncompetitive inhibitors ofCYP2C9enzymeincludenifedipine,tranylcypromine,phenethyl isothiocyanate, and 6-hydroxyflavone.17

Summarywww.bio.miami.eduApplications of Enzyme Kinetics studyOne of the applications of enzyme kinetics is the determination of dissociation constants for antigen-antibody interactions in solution.

Applications of Enzyme Kinetics studyOne 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 Kinetics studyOne 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 enzymes activity is affected by concentration, ph etc studying the kinetics might help in providing suitable conditions for drug to be efficient.alevelnotes.comReferences :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 solutionMarc F. Hoylaerts,Alex Bollen,Marc E. De Broe

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