BCHE 395-14 2013 Chymotrypsin Mechanism

specicity aa o r1 1 r c ch n h trypsin chymotrypsin

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Unformatted text preview: Covalent Catalysis H2O A−B + X: ⟹ A−X + B ⟹ A + X: + B •  Characterized by the existence of a (transient) covalently bound enzyme substrate (ES) intermediate –  Look for a nucleophilic group (X:) on enzymes –  Can be a reac6ve serine, thiolate, amine, or carboxylate (so which amino acids??) •  Criteria to establish nucleophilic catalysis: –  (a) isola6on and characteriza6on of the E- S covalent intermediate –  (b) demonstrate that the isolated E- S intermediate reacts consistent with the observed overall rate 11 Metal Ion Catalysis •  Involves a metal ion bound to the enzyme •  Can be involved in orienta6on of the substrate •  Interacts with substrate to facilitate binding – Stabilizes nega6ve charges •  Par6cipates in oxida6on reac6ons 12 Covalent Catalysis, Acid- Base Catalysis ex. Chymotrypsin •  Serine Protease •  Single polypep6de •  Cleaves carboxyl- side of aroma3c amino acids •  Catalyzes the hydrolysis of the pep3de bond (109) è༎ 13 Chymotrypsin Protease (25 kDa) that cleaves C- side of Phe, Tyr, Trp and hydrolyzes of variety of esters, amides, etc... Specificity AA O R1 1 R C CH N H Trypsin Chymotrypsin Pepsin Elastase C O 2 2 1 2 H N R2 2 Lys, Arg Tyr, Trp, Phe Leu, Phe, Trp, Tyr Ala, Gly, Ser, Val 14 Chymotrypsin 15 Chymotrypsin Trypsin •  Chymotrypsin: produced as an inac6ve zymogen in the pancreas of mammals •  Ac6vated by proteolysis in the small intes6ne to aid in protein diges6on 16 Disulfide bonds Chymotrypsin •  Ac6ve site cataly3c groups shown in red •  Ser 195 •  His 57 •  Asp 102 17 Ac6ve Site of Chymotrypsin So what aas does chymotrypsin recognize with Substrate that would need this hydrophobic pocket? •  Cataly3c triad •  Ser195: nucleophile for covalent catalysis •  His57: general acid/ base catalysis •  Asp102: strong H- bond (LBHB) increases His57 pK to > 12 in ES complex Low- Barrier Hyd...
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