Bio Tech lab report CH 369L - 02/04/09 Experiment # 3...

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02/04/09 Experiment # 3 Biochemical Techniques a) Use of enzyme as reagents; Coupled enzyme assays b) Enzyme kinetics Instructor: Dr. Gene McDonald Introduction: In this experiment, we made determination of enzyme activity and enzyme kinetics. The enzyme is a catalyst that increases the rate of a chemical or biological reaction without affecting the equilibrium of that reaction. In addition, it is not consumed in this reaction. Enzymes always provide its chemical reaction an exceptional specificity and due to its protein nature, their reaction always proceeds at very mild temperature. In the following figure 1, there is a good comparison between a catalyzed and unanalyzed reaction but this catalyst is not an enzyme. There is a clear difference between both reactions as the activation energy (∆G ) of the catalyzed reaction clearly decreased while increasing the rate of reaction. In figure 2, there is a good comparison between an enzyme catalyzed reaction and the corresponding non-enzyme catalyzed reaction. In the catalyzed reaction, when ES complex formed, there is very large amount of binding reaction produced due to some bond formation between the enzyme and its substrate. This produced energy helps to decrease the activation energy of the enzyme catalyzed reaction, which causes reaction rate enhancement of more than 10 6 of the unanalyzed reaction. Figure 1: Comparison between the activation energy of catalyzed and unanalyzed chemical reactions 1 Switzer and Garrity through Dr G. McDonald Lecture notes
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02/04/09 Experiment # 3 Biochemical Techniques Figure 2: Comparison between the activation energy of Enzyme-catalyzed and Non- enzyme-catalyzed chemical reactions Michaelis and Menten are two famous scientists who put the main basics of enzyme kinetics. In any enzymatic reaction, the substrate [S] interacts with the enzyme [E] forming Enzyme/Substrate complex [ES], which is transferred to Enzyme/Product complex [EP] that dissociate finally to the product [P] and the free enzyme [E]. This reaction can be summarized in the following equations that we will use to study the kinetics of this reaction. 2 Switzer and Garrity through Dr G. McDonald Lecture notes E+S ES EP E+P E+S ES E+P k 1 k -1 k 2 v = dP/dt = k [S [ v = dP/dt = k 2 [ES [
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02/04/09 Experiment # 3 Biochemical Techniques Figure 3: Steady state of an enzymatic reaction From the previous equations, we can measure the velocity of product formation using the rate of dissociation of [ES] complex to the product and the free enzyme. At the steady state of any enzymatic reaction (Figure 3), the rate of dissociation of [ES] complex equals the rate of association of this complex, as at this point d[ES]/dt = zero. The rate of dissociation of [ES] complex = k -1 [ES]+k 2 [ES] and the rate of association of [ES] complex = k 1 [E][S]. So at the steady state, k -1 [ES]+k 2 [ES] = k 1 [E] [S]. We can use this relationship to deduce the Michaelis constant using the following equations: If d[ES]/dt = 0 Then
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Bio Tech lab report CH 369L - 02/04/09 Experiment # 3...

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