21 - Lecture 14: Catalytic Mechanisms 2 Monday, August 23,...

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Unformatted text preview: Lecture 14: Catalytic Mechanisms 2 Monday, August 23, 2010 As a model system for a detailed mechanism that an enzyme uses to catalyze a chemical reaction, various proteases illustrate the general principles. Monday, August 23, 2010 The basic reaction that all proteases catalyze is the hydrolysis of a peptide bond. This reaction for chymotrypsin (cleaves after large hydrophobic residues) can be mimicked using an ester compound: Monday, August 23, 2010 The kinetic curve for this reaction catalyzed by chymotrypsin looks like: Monday, August 23, 2010 The presence of a burst phase correlates to the amount of enzyme that is added to the reaction; i.e., it is stoichiometric with the quantity of active enzyme . This suggests that there are TWO steps in the mechanism; a rapid initial step and a slower second step. Monday, August 23, 2010 A second experiment was performed to identify the active site residue using a suicide inhibitorDIPF (diisopropylphosphouoridate): This reagent specifically modified a single serine residue in the enzyme (Ser 195), demonstrating that this amino acid was playing a central role in the catalytic mechanism. Monday, August 23, 2010 Together, these experiments suggested a covalent catalysis mechanism comprising two steps: The first step rapidly liberates the colored product, p-nitrophenolate, while the slower second step was required to reset the enzyme. Monday, August 23, 2010 A second essential residue for the reaction was identified through an anity labeling reaction: The compound TPCK is a substrate mimic, which binds to the active site of the enzyme (it contains a group that looks like Phe). This places a highly reactive chloromethyl keto group that reacts with a single histidine residue (His 57): Thus, prior to a structure of the enzyme, Ser 195 and His 57 had been identified as components of the active site....
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This note was uploaded on 11/12/2010 for the course CHEN 3320 at Colorado.

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21 - Lecture 14: Catalytic Mechanisms 2 Monday, August 23,...

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