chymotrypsin

4 types of irreversible inhibitions

Group specific reagents

Affinity labels/Substrate analogs

Mechanism based inhibitors

TS analogs

Group specific reagents

React w specific functional group DIPF

Affinity labels/substrate analogs

Structurally similar to substrate and have a chemically reactive group

TPCK

Mechanism bases inhibitor

Catalytic mechanism leads to inactivation of enzyme

Penicillin

3 features of active site for catalysis

Substrate specificity

Proximity and orientation effects

TS stabilization

pH rate profile told us

Kcat depends on ionizable group with a pH = 7 which leans to His

Group specific reagents DIPf tells

Serine is importing

DIPF inhibits chymotrypsin by modifying one specific ser - shows how reactive that ser is

Affinity label/substrate analog TPCK tells us

His is present and important

TPCK can bind to chymotrypsin. The TPCK covalently modifies His 57 showing that His is in active site bc TPCK is positioned w specific binding to active site

Pre-steady state kinetics shows us

Covalent catalysis

Catalysis occurs in 2 steps

1. Fast actuation step Ser covalent bond to ache group of substrate

2. Slow deacylation Acyl enzyme intermediate hydrolysis to release carboxylix acid of substrate and regenerates enzyme

Crystal structure tells

Reveals active site residues - catalytic triad

His acts as base to deprotonate Ser

Asp polarizes His to make it a better base

Acid base catalysis

Amino acid side chains can participate in proton transfer reactions

Covalent catalysis

Enzyme forms temporary covalent bond with substrate (acyl-enzyme)

His role

His acts as a base to deprotonate Ser and 2nd substrate water to make them better nucelophiles. It also acts as an acid to proton ate the leaving group after collapse of the tetrahedral intermediate.