BCHE 395-14 2013 Chymotrypsin Mechanism

The amino terminus the carboxy chymotrypsin mechanism

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Unformatted text preview: amino acids? What if the muta6on was a conserved change? 22 Chymotrypsin Mechanism Step 1: Substrate Binding So which end of this “pep6de” is the amino terminus? Which is the carboxy? Covalent Catalysis: Chymotrypsin Mechanism: Free Enzyme Polarized interac6on 102 Asp His57 O LBHB C O His57 pKa change H N N Active site Oxyanion hole READ Fig. 6- 22, pp. 216- 217 H N Gly Ser195 HO 193 H N Ser195 Hydrophobic pocket 24 Chymotrypsin Mechanism: Forma6on of ES Complex Base (B:)à༎ proton acceptor His57 O 102 Asp C His57 acts as a general base, making Ser195 a beler nucleophile O H N N AAn C CH NH C CH NH AAn O R1 O H So which end of this “pep6de” is the amino terminus? Which is the carboxy? Ser195 HO N Gly 193 H N Ser195 25 Chymotrypsin Mechanism Step 2: Nucleophilic Alack Chymotrypsin Mechanism: Forma6on of ES Complex: another view Oxyanion hole VV His57 acts as a general base, making Ser195 a beler nucleophile (Michaelis complex): Trigonal carbonyl carbon 27 Chymotrypsin Mechanism: Forma6on of ES Complex: another view The tetrahedral intermediate occupies the previously unoccupied oxyanion hole VV Intermediate 1: Tetrahedral transi6on state Terrific example of how ac3ve sites are designed to bind to transi3on state (Fig. 6- 5: S3ckase!) 28 Chymotrypsin Mechanism: Transi6on State (TS) 1 includes a covalent intermediate His57 O 102 Asp C O H N N H AAn Ser195 O C CH NH C CH NH AAn O R1 O H N Gly 193 H •  TS breaks down to cleave pep6de bond •  His 57 acts as general acid to protonate the amide nitrogen of the cleaved pep6de bond N Ser195 29 Chymotrypsin Mechanism: Product 1 Released His57 O 102 Asp C AAn O H C CH O N N Pep6de C- terminal to the cleaved bond is released R1 NHH So which end of this “pep6de” is released first? The amino terminus? The carboxy? Ser195 O C CH NH AAn O H Acyl- Enzyme Intermediate N Gly 193 H N Ser195 Product 2 s6ll COVALENTLY bonded to Ser195 30 Chymotrypsin Mechanism Step 3: Substrate Cleavage So which end of this “pep6de” is released first? The amino terminus? The carboxy? Chymotrypsin Mechanism: Hydrolysis step O 102 Asp C His57 acts as general base to ac6vate water for alack His57 O H N N H H O Ser195 O Ester linkage C CH NH AAn O Acyl- Enzyme Intermediate H N Gly 193 H N Ser195 32 Chymotrypsin Mechanism Step 4: Water Comes In Chymotrypsin Mechanism Step 5: Water Alacks Chymotrypsi...
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