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Catalytic Mechanism of Chymotrypsin slide 1

Catalytic Mechanism of Chymotrypsin slide 1. Chymotrypsin Protease: catalyze hydrolysis of proteins in small intestine Specificity: Peptide bond on carboxyl side of aromatic side chains (Y, W, F) & Large hydrophobic residues (Met,…). Three polypeptide chains cross-linked to each other

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Catalytic Mechanism of Chymotrypsin slide 1

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  1. Catalytic Mechanism of Chymotrypsinslide 1 • Chymotrypsin • Protease: catalyze hydrolysis of proteins in small intestine • Specificity: Peptide bond on carboxyl side of aromatic side chains (Y, W, F) & Large hydrophobic residues (Met,…) • Three polypeptide chains cross-linked to each other • Three catalytic residues: Ser195, His57, & Asp102

  2. Catalytic Mechanism of Chymotrypsinslide 2

  3. Catalytic Mechanism of Chymotrypsinslide 2

  4. Catalytic Mechanism of Chymotrypsinslide 3

  5. Catalytic Mechanism of Chymotrypsin slide 4

  6. Summary for the Catalytic Mechanism of Chymotrypsin • Mechanism • General acid-base catalysis & Covalent catalysis • Two steps: Acylation & Deacylation (rate limiting; reverse of acylation with water substituting the amine component) • Key features • Active Ser195& roles of the three catalytic residues • Tetrahedral transition state • OxyanionandOxyanion hole • Acyl-enzyme intermediate

  7. Chymotrypsin & elastase main chain conformation (superimposed) Serine Protease Family • Serine Proteases • Chymotrypsin • Trypsin • Elastase • Similarity • Similar 3D structure • Catalytic triad • Oxyanion hole • Covalent acyl-enzyme intermediate • Secreted by pancrease as inactive precursors

  8. Specificity Difference of Chymotrypsin, Trypsin, and Elastase • Substrate specificity • Chymotrypsin: aromatic or bulky nonpolar side chain • Trypsin: Lys or Arg • Elastase: smaller & uncharged side chains • Small structural difference in the binding site explains the substrate specificity • nonpolar pocket • no pocket present • as two Gly in chymotrypsin • are replaced by Val and Thr • Asp (negatively charged) • vs. Ser in Chymotrypsin

  9. Carboxypeptidase A • Digestive enzyme • Hydrolyzes carboxyl terminal peptide bond • Prefer bulky and aliphatic residues • 3D structure • Single polypeptide (307 amino acids) •  helices (38%) and  (17%) (compact, ellipsoid) A tightly bound Zn2+ Essential for catalysis Coordinated to 1H2O, 2 His, 1 Glu

  10. Substrate Binding Induces Large Structural Changes at the Active Site

  11. Substrate Binding Induces Large Structural Changes at the Active Site • 3D Structure of peptidase A/glycyltyrosine complex • Substrate-inducedstructural change at active site • 12 Åmovement of Tyr248-OH & rotation(Moves from surface to substrate terminal COO-) • New interaction: Tyr248 OH  –OC=O • Closes active-site cavity • Extrude water from cavity • Arg145 moves 2 Å • New interaction: Arg145 & –OC=O (substrate) • Terminal side chain of substrate • Now sits in a hydrophobic pocket • Induced-fit model (Daniel Koshland, Jr.)

  12. Substrate Binding at the Active Site

  13. Catalytic Mechanism of Carboxypeptidase A • The H2O molecule is activated by • Bound Zn2+ andCOO– of Glu270 • ActivatedH2Oattacks the C=O group of the scissile peptide bond • Glu270 simultaneously accepts a H+ from H2O • A negatively chargedtetrahedral intermediate is formed • Intermediate is stabilized by Zn2+ and Arg127 • H+ transfer from COOH of Glu270 to the peptide NH • Peptide bond is concomitantly cleaved • The reaction products diffuse away • Summary: • Activation of H2O by Zn2+ and Glu270 • Proton abstraction and donation by Glu270 • Electrostatic stabilization of tetrahedral intermediate by Arg127 and Zn2+

  14. Catalytic Mechanism of Carboxypeptidase A

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