3/7/201711Principles of Enzyme CatalysisPrinciples of Enzyme CatalysisI)Enzyme KineticsII)Inhibition of Enzyme ActivityIII)Second-Order ReactionsIV)Mechanisms of Enzyme ActionV)Example: Serine ProteasesVI)Enzyme Regulation2I)Enzyme KineticsMichaelis-Menten equation can be used as long as:•[E] is constant•Formation of EAcomplex. Breakdown of EAto form products is assumed to be slower than (i) formation of EA and (ii) breakdown of EAto re-form Eand A. Vmax= k3[Et]•Steady-state: concentration of EAcomplex is constant (assumption 1)•Only the initial rate V0is measured (k4can be ignored, no inhibition or reversibility -assumption 2)•[A] >> [E] and Aconcentration is considered constant (assumption 3)𝑉0=𝑉𝑚𝑎𝑥[?]𝐾𝑚+ [?]𝐸 + ?𝑘1𝑘2𝐸?𝑘3𝑘4𝐸 + ?31)Mix enzymeand substrateand follow the reaction2)Determine V0at different substrate concentrations3)Make plots of V0vs. [A]or 1/V0vs. 1/[A]Enzyme Assay4
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