CHYMOTRYPSIN INTRO MISSION: STRATEGY: PROFILE: RELATIVES: SUSPECTS: 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: PROFILE: Protease Secreted by pancreas 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: acylation of the substrate to form an acyl-enzyme intermediate Steady-state phase: deacylation in order to return the enzyme to its original state (following Michaelis-Menten kinetics) KEY FEATURES: Tetrahedral transition state Oxanion hole Acyl-enzyme intermediate

1 CHYMOTRYPSIN PREPARING FOR ATTACK FRIENDLY FIRE TACTICS STAGE 1: 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)

2 CHYMOTRYPSIN READY…SET…CHARGE! TACTICS STAGE 1: 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 oxyanion hole stabilizes the negative charge on the carbonyl

3 CHYMOTRYPSIN RELIEVING THE TENSION Transition state collapses 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”

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

5 CHYMOTRYPSIN FINALLY! INTRUDER ALERT TACTICS STAGE 2: 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”)

6 CHYMOTRYPSIN RELIEVING THE TENSION (AGAIN) 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”)

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

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