03 enzyme kinetics

03 enzyme kinetics - CEE 266 ENVIRONMENTAL BIOTECHNOLOGY...

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Unformatted text preview: CEE 266 ENVIRONMENTAL BIOTECHNOLOGY Lecture 3 (Enzyme Kinetics: Michaelis-Menten equations, Lineweaver-Burk plots, inhibition) Enzymes   Biological catalysts – increase the rate of a thermodynamicallyfavorable reaction   Literally “in yeast”   Proteins or RNA   Substrate specificity   Regenerated at the end of reaction Six Classes of Enzymes Enzymes Need Support Enzyme activity requires the native 3D structure, and often the presence of a cofactor such as a metal ion or prosthetic group. Apoenzymes lack the prosthetic group; holoenzymes contain it and are fully functional. Activation Energy and Catalysis Figure 5.6 Catalysis and Enzymes   Two mechanisms of catalysis Enzyme-Substrate Binding Michaelis-Menten Kinetics   Rate of reaction depends on   Enzyme characteristics   Substrate Concentration   Affinity of enzyme for substrate   Formation of an enzyme-substrate complex   The ES complex is in rapid equilibrium with free enzyme   Breakdown of ES to form products is assumed to be slower than   formation of ES   breakdown of ES to re-form E and S Michaelis-Menten Kinetics Steady State v = k2[ES], if this is the rate-limiting step* [Enzyme]total = [E]t = [E] + [ES] 1. Assume equilibrium, if k-1 >> k2: KS = k-1/k1 = [E][S]/[ES] or 2. Assume steady state: d[ES]/dt = 0 [*Note: v is always measured as an initial rate!] Michaelis-Menten Kinetics Michaelis-Menten Kinetics Understanding Km The ”affinity constant"   Km is a specific substrate concentration   Km is a constant derived from rate constants   Km is, under true Michaelis-Menten conditions, an estimate of the dissociation constant of E from S   Small Km means tight binding; high Km means weak binding Understanding Vmax The theoretical maximal velocity   Vmax is a rate constant   Vmax is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality   To reach Vmax would require that ALL enzyme molecules are tightly bound with substrate   Vmax is asymptotically approached as substrate is increased Dual Order Kinetics Combination of 0-order and 1st-order kinetics   When S is low, the equation for rate is 1st order in S   When S is high, the equation for rate is 0-order in S   The Michaelis-Menten equation describes a rectangular hyperbolic dependence of v on S! Double Reciprocal Plot Turnover Number Examples of Turnover Numbers Enzyme Affinity for Substrates Catalytic Efficiency, kcat/Km => “Perfect enzyme” Diffusion-controlled limit: 108-109 M-1s-1   An estimate of "how perfect" the enzyme is   kcat/Km is an apparent second-order rate constant   It measures how the enzyme performs when S is low   The upper limit for kcat/Km is the diffusion limit - the rate at which E and S diffuse together Types of Enzyme Inhibition Enzyme Inhibition Enzyme Inhibition Enzyme Inhibition Enzyme Inhibition Enzyme Inhibition Enzyme Inhibition Summary Example 1 Michaelis-Menten kinetics can be used to describe the dependence of biochemical reaction rate,V, on the concentration of substrate S. a)  For Vmax = 22 day-1 and Km = 10-6 M, what is the growth rate at a substrate concentration of 10-7 M. Example 1b b)  If S were 10 times larger, how would this change your answer quantitatively? c)  If S were 1000 times larger, how would this change your answer quantitatively? Please explain why, mechanistically, this would be the case. Example 1c If S were 1000 times larger, how would this change your answer quantitatively? Please explain why, mechanistically, this would be the case. Answers: V = 2 day-1, @10*S, V = Vmax/2 = 11 day-1, at 1000*S, V ~ Vmax = 22 day-1. ...
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This note was uploaded on 02/02/2012 for the course CEE 266 taught by Professor Shailymahendra during the Fall '11 term at UCLA.

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