catalytic mechanism of chymotrypsin slide 1 chymotrypsin –protease: catalyze hydrolysis of...
Post on 21-Dec-2015
233 views
TRANSCRIPT
![Page 1: Catalytic Mechanism of Chymotrypsin slide 1 Chymotrypsin –Protease: catalyze hydrolysis of proteins in small intestine –Specificity: Peptide bond on carboxyl](https://reader030.vdocuments.net/reader030/viewer/2022020800/56649d6a5503460f94a47b5f/html5/thumbnails/1.jpg)
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
![Page 2: Catalytic Mechanism of Chymotrypsin slide 1 Chymotrypsin –Protease: catalyze hydrolysis of proteins in small intestine –Specificity: Peptide bond on carboxyl](https://reader030.vdocuments.net/reader030/viewer/2022020800/56649d6a5503460f94a47b5f/html5/thumbnails/2.jpg)
Catalytic Mechanism of Chymotrypsin slide 2
![Page 3: Catalytic Mechanism of Chymotrypsin slide 1 Chymotrypsin –Protease: catalyze hydrolysis of proteins in small intestine –Specificity: Peptide bond on carboxyl](https://reader030.vdocuments.net/reader030/viewer/2022020800/56649d6a5503460f94a47b5f/html5/thumbnails/3.jpg)
Catalytic Mechanism of Chymotrypsin slide 2
![Page 4: Catalytic Mechanism of Chymotrypsin slide 1 Chymotrypsin –Protease: catalyze hydrolysis of proteins in small intestine –Specificity: Peptide bond on carboxyl](https://reader030.vdocuments.net/reader030/viewer/2022020800/56649d6a5503460f94a47b5f/html5/thumbnails/4.jpg)
Catalytic Mechanism of Chymotrypsin slide 3
![Page 5: Catalytic Mechanism of Chymotrypsin slide 1 Chymotrypsin –Protease: catalyze hydrolysis of proteins in small intestine –Specificity: Peptide bond on carboxyl](https://reader030.vdocuments.net/reader030/viewer/2022020800/56649d6a5503460f94a47b5f/html5/thumbnails/5.jpg)
Catalytic Mechanism of Chymotrypsin slide 4
![Page 6: Catalytic Mechanism of Chymotrypsin slide 1 Chymotrypsin –Protease: catalyze hydrolysis of proteins in small intestine –Specificity: Peptide bond on carboxyl](https://reader030.vdocuments.net/reader030/viewer/2022020800/56649d6a5503460f94a47b5f/html5/thumbnails/6.jpg)
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
![Page 7: Catalytic Mechanism of Chymotrypsin slide 1 Chymotrypsin –Protease: catalyze hydrolysis of proteins in small intestine –Specificity: Peptide bond on carboxyl](https://reader030.vdocuments.net/reader030/viewer/2022020800/56649d6a5503460f94a47b5f/html5/thumbnails/7.jpg)
Serine Protease FamilyChymotrypsin & 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
![Page 8: Catalytic Mechanism of Chymotrypsin slide 1 Chymotrypsin –Protease: catalyze hydrolysis of proteins in small intestine –Specificity: Peptide bond on carboxyl](https://reader030.vdocuments.net/reader030/viewer/2022020800/56649d6a5503460f94a47b5f/html5/thumbnails/8.jpg)
Specificity Difference of Chymotrypsin, Trypsin, and Elastase
nonpolar pocket Asp (negatively charged) vs. Ser in Chymotrypsin
no pocket present as two Gly in chymotrypsin are replaced by Val and Thr
• 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
![Page 9: Catalytic Mechanism of Chymotrypsin slide 1 Chymotrypsin –Protease: catalyze hydrolysis of proteins in small intestine –Specificity: Peptide bond on carboxyl](https://reader030.vdocuments.net/reader030/viewer/2022020800/56649d6a5503460f94a47b5f/html5/thumbnails/9.jpg)
Carboxypeptidase A
A tightly bound Zn2+ Essential for catalysis
Coordinated to 1 H2O, 2 His, 1 Glu
• 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)
![Page 10: Catalytic Mechanism of Chymotrypsin slide 1 Chymotrypsin –Protease: catalyze hydrolysis of proteins in small intestine –Specificity: Peptide bond on carboxyl](https://reader030.vdocuments.net/reader030/viewer/2022020800/56649d6a5503460f94a47b5f/html5/thumbnails/10.jpg)
Substrate Binding Induces Large Structural Changes at the Active Site
![Page 11: Catalytic Mechanism of Chymotrypsin slide 1 Chymotrypsin –Protease: catalyze hydrolysis of proteins in small intestine –Specificity: Peptide bond on carboxyl](https://reader030.vdocuments.net/reader030/viewer/2022020800/56649d6a5503460f94a47b5f/html5/thumbnails/11.jpg)
• 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 OH –OC=O
– Closes active-site cavity
– Extrude water from cavity
• Arg145 moves 2 Å– New interaction: Arg145 & –OC=O (substrate)
• Terminal side chain of substrate
– Now sits in a hydrophobic pocket
– Induced-fit model (Daniel Koshland, Jr.)
Substrate Binding Induces Large Structural Changes at the Active Site
![Page 12: Catalytic Mechanism of Chymotrypsin slide 1 Chymotrypsin –Protease: catalyze hydrolysis of proteins in small intestine –Specificity: Peptide bond on carboxyl](https://reader030.vdocuments.net/reader030/viewer/2022020800/56649d6a5503460f94a47b5f/html5/thumbnails/12.jpg)
Substrate Binding at the Active Site
![Page 13: Catalytic Mechanism of Chymotrypsin slide 1 Chymotrypsin –Protease: catalyze hydrolysis of proteins in small intestine –Specificity: Peptide bond on carboxyl](https://reader030.vdocuments.net/reader030/viewer/2022020800/56649d6a5503460f94a47b5f/html5/thumbnails/13.jpg)
Catalytic Mechanism of Carboxypeptidase A• The H2O molecule is activated by
– Bound Zn2+ and COO– of Glu270• Activated H2O attacks the C=O 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+
![Page 14: Catalytic Mechanism of Chymotrypsin slide 1 Chymotrypsin –Protease: catalyze hydrolysis of proteins in small intestine –Specificity: Peptide bond on carboxyl](https://reader030.vdocuments.net/reader030/viewer/2022020800/56649d6a5503460f94a47b5f/html5/thumbnails/14.jpg)
Catalytic Mechanism of Carboxypeptidase A