Enzyme kinetics

enzyme kinetics
michealis menton analysis,
eadie analysis
lineweaver analysis

1. Enzyme Kinetics SAKHARKAR MOHAMMAD ANZAR R.J. COLLEGE MSC-I SEM-Ii 2013 PHYSICAL CHEMISTRY 1 2/13/2013 By Mohd Anzar Sakharkar

2. Enzymes Enzymes are proteins which acts as biocatalyst. It alters the rate of reaction in biological process. 2/13/2013 2 By Mohd Anzar Sakharkar

3. Enzyme action Like all catalysts, enzymes accelerate the rates of reactions while experiencing no permanent chemical modification as a result of their participation. Enzymes can accelerate, often by several orders of magnitude, reactions that under the mild conditions of cellular concentrations, temperature, p H, and pressure would proceed imperceptibly in the absence of the enzyme. 2/13/2013 3 By Mohd Anzar Sakharkar

4. Enzyme Kinetics 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 is investigated. Studying an enzyme's kinetics in this way can reveal the catalytic mechanism of this enzyme 2/13/2013 4 By Mohd Anzar Sakharkar

5. 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. These mechanisms can be divided into single-substrate and multiple-substrate mechanisms. Kinetic studies on enzymes that only bind one substrate, such as triosephosphate isomerase , aim to measure the affinity with which the enzyme binds this substrate and the turnover rate. 2/13/2013 5 By Mohd Anzar Sakharkar

6. Michealis-Menten Analysis Michaelis–Menten kinetics is one of the simplest and best-known models of enzyme kinetics. The model serves to explain how an enzyme can cause kinetic rate enhancement of a reaction and why the rate of a reaction depends on the concentration of enzyme present.  2/13/2013 6 By Mohd Anzar Sakharkar

7. To begin our discussion of enzyme kinetics, let's define the number of moles of product (P) formed per time as  V . The variable,  V,  is also referred to as the rate of catalysis of an enzyme. For different enzymes,  V  varies with the concentration of the substrate, S. At low S,  V  is linearly proportional to S, but when S is high relative to the amount of total enzyme,   V  is independent of S. 2/13/2013 7 By Mohd Anzar Sakharkar

8. To understand Michaelis-Menten Kinetics, we will use the general enzyme reaction scheme shown below, which includes the back reactions in addition the forward reactions: The table below defines each of the rate constants in the above scheme.   2/13/2013 8 By Mohd Anzar Sakharkar

9. The table below defines each of the rate constants in the above scheme.    Rate Constant            Reaction k 1 The binding of the enzyme to the substrate forming the enzyme substrate complex. k 2 The dissociation of the enzyme-substrate complex to free enzyme and substrate . k 3   Catalytic rate; the catalysis reaction producing the final reaction product and regenerating the free enzyme. This is the rate limiting step. k 4  The reverse reaction of catalysis . 2/13/2013 9 By Mohd Anzar Sakharkar

10. Substrate Complex The ES complex is formed by combining enzyme E with substrate S at rate constant k 1 .  The ES complex can either dissociate to form E F  (free enzyme) and S, or form product P at rate constant k 2  and k 3 , respectively. 2/13/2013 10 By Mohd Anzar Sakharkar

11. The velocity equation can be derived following method: The rates of formation and breakdown of the E - S complex are given in terms of known quantities: The rate of formation of E-S =  (with the assumption that [P] =0) The rate of breakdown of E-S =   =  2/13/2013 11 By Mohd Anzar Sakharkar

12. At steady state,  = Therefore, rate of formation of E-S = rate of breakdown of E-S So,  Dividing through by k1: [E] [S] =    [E-S] Substituting    with k M : k M  =  2/13/2013 12 By Mohd Anzar Sakharkar

13.   implies that half of the active sites on the enzymes are filled. Different enzymes have different    values.  They typically range from 10 -1  to 10 -7  M.  The factors that affect     are: pH temperature ionic strengths the nature of the substrate 2/13/2013 13 By Mohd Anzar Sakharkar

14. Substituting [E F ] with [E T ]-[ES]:                 E T  = [ES] + [E F ] ([E T ] - [ES]) [S] = k M  [ES] [E T ] [S] -[ES][S] = k M  [ES] [E T ] [S] = [ES] [S] + k M  [ES] [E T ] [S] = [ES] ([S] + k M ) Solving for [ES]:  [ES] =  2/13/2013 14 By Mohd Anzar Sakharkar

15. The rate equation from the rate limiting step is: Vo  =    = k 2 [ES] Multiplying both sides of the equation by k2: k 2  [ES] =  Vo  = When S>>K M,   v o  is approximately equal to k 2 [E T ]. When the [S] great, most of the enzyme is found in the bound state ([ES]) and V o  = V max We can then substitue k 2 [E T ] with V max   to get the  Michaelis Menten Kinetic Equation: v o  = 2/13/2013 15 By Mohd Anzar Sakharkar

16. Lineweaver -Burk Plot The  Lineweaver –Burk plot  is a graphical representation of the Lineweaver –Burk equation of enzyme kinetics, described by Hans Lineweaver  and Dean Burk in 1934. 2/13/2013 16 By Mohd Anzar Sakharkar

17. Derivation The plot provides a useful graphical method for analysis of the  Michaelis-Menten  equation: Taking the reciprocal gives V  is the reaction velocity (the reaction rate) K m  is the  Michaelis–Menten constant V max  is the maximum reaction velocity [ S ] is the substrate concentration 2/13/2013 17 By Mohd Anzar Sakharkar

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19. Apply this to equation for a straight line   and we have: When we plot    versus  , we obtain a straight line.   2/13/2013 19 By Mohd Anzar Sakharkar

20. The Lineweaver –Burk plot was widely used to determine important terms in enzyme kinetics, such as  K m  and  V max , before the wide availability of powerful computers and non-linear regression software. The  y -intercept of such a graph is equivalent to the inverse of  V max ; the  x -intercept of the graph represents −1/ K m . It also gives a quick, visual impression of the different forms of enzyme inhibition. The double reciprocal plot distorts the error structure of the data, and it is therefore unreliable for the determination of enzyme kinetic parameters. 2/13/2013 20 By Mohd Anzar Sakharkar

21. Eadie–Hofstee diagram Eadie–Hofstee diagram is a graphical representation of enzyme kinetics in which reaction velocity is plotted as a function of the velocity vs. substrate concentration ratio: V =reaction velocity K m  =  Michaelis–Menten constant [ S ] = substrate concentration V max  = maximum reaction velocity. 2/13/2013 21 By Mohd Anzar Sakharkar

22. It can be derived from the Michaelis–Menten equation as follows: invert and multiply with  : Rearrange: Isolate v: 2/13/2013 22 By Mohd Anzar Sakharkar

23. A plot of v vs v/[S] will yield  V max  as the y-intercept,  V max /K m  as the x-intercept, and  K m  as the negative slope. Like other techniques that linearize the  Michaelis–Menten equation, the Eadie-Hofstee plot was used historically for rapid identification of important kinetic terms like  K m  and  V max , but has been superseded by nonlinear regression methods that are significantly more accurate and no longer computationally inaccessible. It is also more robust against error-prone data than the  Lineweaver –Burk plot, particularly because it gives equal weight to data points in any range of substrate concentration or reaction velocity. Both plots remain useful as a means to present data graphically. 2/13/2013 23 By Mohd Anzar Sakharkar

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25. BIBILIOGRAPHY Atkins, Peter and de Paula, Julio.  Physical Chemistry for the Life Sciences . New York, NY: W. H. Freeman and Company, 2006. Page 309-313. Stryer , Lubert .  Biochemistry (Third Edition) . New York, NY: W.H. Freeman and Company, 1988. Page 187-191. Chang, Raymond.  Physical Chemistry for the Biosciences . Sansalito , CA: University Science, 2005. Page 363-371. 2/13/2013 25 By Mohd Anzar Sakharkar