Catalytic Mechanism of Chymotrypsin slide 1

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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
• Three catalytic residues Ser195, His57, Asp102

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Catalytic Mechanism of Chymotrypsin slide 2
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Catalytic Mechanism of Chymotrypsin slide 2
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Catalytic Mechanism of Chymotrypsin slide 3
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Catalytic Mechanism of Chymotrypsin slide 4
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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
• Oxyanion and Oxyanion hole
• Acyl-enzyme intermediate

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Serine Protease Family
Chymotrypsin elastase main chain
conformation (superimposed)

• Serine Proteases
• Chymotrypsin
• Trypsin
• Elastase
• Similarity
• Similar 3D structure

• Catalytic triad
• Oxyanion hole
• Covalent acyl-enzyme intermediate
• Secreted by pancrease as inactive precursors

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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
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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 1 H2O, 2 His, 1 Glu
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Substrate Binding Induces Large Structural
Changes at the Active Site
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Substrate Binding Induces Large Structural
Changes at the Active Site

• 3D Structure of peptidase A/glycyltyrosine
  complex
• Substrate-induced structural change at active
  site
• 12 Å movement of Tyr248-OH rotation (Moves from
  surface to substrate terminal COO-)
• New interaction Tyr248 ?O?H ??? O?CO
• Closes active-site cavity
• Extrude water from cavity
• Arg145 moves 2 Å
• New interaction Arg145 O?CO (substrate)
• Terminal side chain of substrate
• Now sits in a hydrophobic pocket
• Induced-fit model (Daniel Koshland, Jr.)

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Substrate Binding at the Active Site
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Catalytic Mechanism of Carboxypeptidase A

• The H2O molecule is activated by
• Bound Zn2 and COO of Glu270
• Activated H2O attacks the CO group of the
  scissile peptide bond
• Glu270 simultaneously accepts a H from H2O
• A negatively charged tetrahedral 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

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Catalytic Mechanism of Carboxypeptidase A