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lecture_16 - Enzyme Kinetics and Catalysis Serine proteases...

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Enzyme Kinetics and Catalysis 3/19/2003
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Serine proteases Diverse and widespread proteolytic enzymes Involved in digestion, development, clotting, inflammation… Common catalytic mechanism
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Use of an Artificial Substrate P-Nitrophenolate is very yellow while the acetate is colorless. This is an example of an artificial substrate!
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The kinetics show 1. A “ burst phase ” where the product is rapidly formed with amounts stoichiometric with the enzyme. 2. Slower steady state that is independent of substrate concentration.
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A covalent bond between a Serine and the substrate suggests an “active Serine”. These Serines can be labeled with inhibitors such as diidopropyl phosphofluoridate specifically killing the enzyme . Ser 195 is specifically labeled
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DIPF is extremely toxic because other active Serines can be labeled. Such as acetylcholine esterase. Nerve gases, serin gas, are very toxic!! Many insecticides also work this way.
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Affinity labeling His 57 is a second important catalytic residue. A substrate containing a reactive group binds at the active site of the enzyme and reacts with a nearby reactive amino acid group. A Trojan horse effect. Tosyl-L-phenylalanine chloromethyl ketone (TPCK)
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The reaction of TPCK with His 57 of chymotrypsin
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Bovine Trypsin
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The catalytic triad
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Bovine trypsin catalytic triad
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Catalytic mechanism 1. After the substrate binds Ser 195 nucleophilically attacks the scissile peptide bond to form a transition state complex called the tetrahedral intermediate (covalent catalysis) the imidazole His 52 takes up the proton Asp 102 is hydrogen bonded to His 57. Without Asp 102 the rate of catalysis is only 0.05% of wild-type. 2. Tetrahedral intermediate decomposes to the acyl- enzyme intermediate. His 57 acts as an acid donating a proton. 3. The enzyme is deacylated by the reverse of step 1 with water the attacking nucleophile and Ser 195 as the leaving group.
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intermediate and causes the carboxyl to move close to the oxyanion hole 2. Now it forms two hydrogen bonds with the enzyme that cannot form when the carbonyl is in its normal conformation. 3. Distortion caused by the enzyme binding allows the
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This note was uploaded on 11/11/2011 for the course CHEM 340 taught by Professor Edwards during the Fall '11 term at BYU.

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lecture_16 - Enzyme Kinetics and Catalysis Serine proteases...

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