Chymotrypsin Lab Report

The Kinetics of α-Chymotrypsin

Introduction

Chymotrypsin is a protease which cleaves proteins by a hydrolysis reaction, it does this by adding a molecule of water to a peptide bond. Although the hydrolysis reaction is thermodynamically favoured in the absence of a catalyst the half-life for a typical hydrolysis reaction by a protease is between 10 and 100 years, needless to say it is extremely slow1. Though this is true peptide bonds are hydrolysed within milliseconds in the body in the presence of catalysts. The kinetic stability of chymotrypsin which gives it hydrolysis resistance is due to the resonance structure that accounts for the planarity of the peptide bond. Such is the strength of this resonance structure that it confers partial
The ester group of the acyl-enzyme is then hydrolysed by nucleophilic attack of serine on the peptide carbonyl group, collapse of the tetrahedral intermediate and the release of the amine component. Histidine 57 then acts as a general acid catalyst and draws a proton away from the water molecule. The OH- ion then attacks the carbonyl carbon atom of the acyl group which forms another tetrahedral intermediate which breaks down to form the carboxylic acid product. The release of this acid then frees up the enzyme to continue
The Folin assay overestimated the chymotrypsin concentration because of the difference in the properties of BSA and chymotrypsin. The different amino acid composition could have had an effect on how much the Folin reagent was reduced which causes the observed colour change. Too many of these amino acids which have this reducing ability in chymotrypsin could have led to the higher observed concentration seen in this