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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
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
slide6

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
serine protease family

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
specificity difference of chymotrypsin trypsin and elastase
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
carboxypeptidase a
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

slide11

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.)
catalytic mechanism of carboxypeptidase a
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+