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CHYMOTRYPSIN

INTRO. CHYMOTRYPSIN. MISSION:. To cleave peptide amide bonds of protein in small intestine. Cleave carboxyl terminus of large nonpolar or aromatic side chains i.e. Tyr, Trp , Phe , and Met Initial general acid-base catalysis Covalent nucleophilic catalysis. STRATEGY:. Protease

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CHYMOTRYPSIN

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  1. INTRO CHYMOTRYPSIN MISSION: To cleave peptide amide bonds of protein in small intestine • Cleave carboxyl terminus of large nonpolar or aromatic side chains i.e. Tyr, Trp, Phe, and Met • Initial general acid-base catalysis • Covalent nucleophilic catalysis STRATEGY: • Protease • Secreted by pancreas PROFILE: RELATIVES: Trypsin, elastase • Aromatic • Large nonpolar • Positive charge • Small nonpolar SUSPECTS: Asp 102, His 57, Ser 195; aka the “catalytic triad” A potent alliance that work alongside each other SITE OF ACTION: Hydrophobic pocket ETA: 16x normal rate Chymotrypsin cleaves peptide bonds by attacking the unreactive carbonyl group with a powerful nucleophile, the Ser 195 residue, which briefly becomes covalently bonded to the substrate, forming an enzyme-substrate intermediate SYNOPSIS: TACTICS: Two part staged plan Initial “burst” phase: acylationof the substrate to form an acyl-enzyme intermediate Steady-state phase: deacylationin order to return the enzyme to its original state (following Michaelis-Menten kinetics) KEY FEATURES: • Tetrahedral transition state • Oxanion hole • Acyl-enzyme intermediate

  2. TACTICS CHYMOTRYPSIN STAGE 1: 1 PREPARING FOR ATTACK Acid base equilibria FRIENDLY FIRE Asp 102 attacks its comrade His 57 via H bonding which steals a H+ from Ser 195 Ser 195 is now ANGRY and a powerful NUCLEOPHILE (an alkoxide anion)

  3. TACTICS CHYMOTRYPSIN STAGE 1: 2 READY…SET…CHARGE! Attack on the substrate Powerful Ser 195 attacks the substrate where it hurts: at the weak carbonyl carbon, an easy target A temporary tetrahedral transition state is formed, an unstable condition Reinforcement: the oxyanionhole stabilizes the negative charge on the carbonyl

  4. TACTICS CHYMOTRYPSIN STAGE 1: 3 RELIEVING THE TENSION Transition state collapses Transition state collapses A stable acyl-enzyme intermediate is formed The substrate carbonyl is attached to the Ser 195 side chain Which is a covalent bond to the active site, hence this is known as “covalent catalysis”

  5. TACTICS CHYMOTRYPSIN STAGE 1: 4 RETREAT! RETREAT! Amino side of the substrate leaves the scene See ya! The remainder of the substrate, an amine product, leaves the active site

  6. TACTICS CHYMOTRYPSIN FINALLY! STAGE 2: 5 INTRUDER ALERT Water enters the scene Water enters the scene His 57 interacts with water, causing it to be polarized Water attacks the acyl-enzyme’s carbonyl in a nucleophilic attack (“nucleophilic catalysis”)

  7. TACTICS CHYMOTRYPSIN STAGE 2: 6 RELIEVING THE TENSION (AGAIN) Transition state collapses Transition state collapses (again) Another tetrahedral transition state is formed (again) The oxyanion hole stabilizes the negative charge (again) The tetrahedral transition state collapses (again) (Notice the theme of “again”)

  8. TACTICS CHYMOTRYPSIN STAGE 2: the end. 7 RETREAT! RETREAT!(again) The substrate leaves the scene Adios! The substrate, a carboxylate product, leaves the active site

  9. Created by STEPHANIE LEUNG • Class of 2013 • Note: the material covered on exams or in lecture may have changed, so I apologize if some of this is no longer relevant. Also, while much of this is my own work, the images and some of the text may have been copied from other sources. I do not claim it as my own. Lastly, I apologize if any of my material is incorrect/inaccurate. I’m just a student myself :) But please do let me know if there are any discrepancies so I can correct them. Thanks! • Questions/comments: please contact me at sleung3@uic.edu

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