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Mechanism of Enzyme Action of Chymotrypsin

Posted on Feb 08 2013 by Sashi Kant, Dale Hancock

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Experiment Overview

Chymotrypsin is a proteolytic enzyme which is synthesised in the pancreas and released into the digestive tract as an inactive proenzyme or zymogen. Once in the gut, it is activated by the removal of part of the peptide chain. Its biological role is to hydrolyse dietary protein in the small intestine. It cleaves the peptide bond on the C (carbonyl) side of the aromatic residues, phenylalanine, tyrosine and tryptophan, and some large hydrophobic residues e.g. methionine and leucine.

 

When studying chymotrypsin’s mechanism of action, an artificial substrate is usually used. There are a number of such substrates such as the methyl esters of amino acid analogs. These substrates are similar to some of chymotrypsin’s biological substrates (it works on esters as well as proteins) and these artificial substrates have the added advantage of producing a coloured product. This enables us to monitor reactions spectrophotometrically.

The students will explore the two-step reaction mechanism, the enzyme dependence of this reaction, the activation of the zymogen, chymotrypsinogen by trypsin.

In this laboratory session the students are going to investigate the mechanism of action of chymotrypsin using the artificial substrate p-nitrophenylacetate  (p-NPA).

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