1·Chemical mechanism of chymotrypsin. a) What are the substrates and the product

Question

1·Chemical mechanism of chymotrypsin. a) What are the substrates and the products of chymotrypsin? Why do you think artificial substrates with brightly colored C-terminal or N-terminal labels are commonly used to study this enzyme? b) What is meant by saying that catalysis by chymotrypsin shows an "early burst phase" and what is the significance of that observation for the mechanism by which chymotrypsin cleaves a protein substrate. c) What are the main roles of the aspartic acid and the histidine in the catalytic triad in chymotrypsin's active site? d) Chymotrypsin is a digestive enzyme produced by the pancreas. Why is chymotrypsin not active inside the pancreas? In other words, what happens in the digestive tract to activate this protease? e) Chymotrypsin uses an activated Ser to cut its substrate proteins. Name some of the other activated groups used by proteases to cut proteins

Explanation / Answer

a. Chymotripsin is a digestive enzyme (protease) that catalyses the breakdown of protein. Therefore the substrates are polypeptide chains, and the products are amino acids. Chymotrypsin, more specifically, catalyses the cleavage of peptide bonds involving L-isomers of tyrosine, phenylalanine, and tryptophan. As chymotrypsin helps in cleavage of peptide bonds resulting in right and left handed fragments of substrate, the brightly coloured labels of N and C terminal help track binding and release events as the reaction proceeds.

b. Early burst phase can be interpreted on a milisecond time scale upon rapid increase in first product concentration due to rapid catalysis. This phenomenon is seen when chymotrypsin catalyses the protein breakdown during digestion. As the first product of the two is rapidly released, but the second product is relatively slowly released, the other chemical cycles that require the second product are slowed down to the rate of formation of second product. This is relatively slow, and is observable in the chymotrypsin mechanism.

c.Histidine or His is a base group containing amino acid which has a tendency to accept proton.Hence Histidine polarises Serine or ser amino acid by accepting proton from its hydroxyl group. At the same time Aspartic Acid or Asp is an acid group containing amino acid having a tendency to loose proton, hence is negatively charged . So Asp stabilizes the protonated Histidine through hydrogen bonding and electrostatics. Hence the three side chains of Chymotrypsin work together to generate a reactive alkoxide anion of Serine which in turn react with the substrate.

d.Pancreas produces a basic form of chymotrypsin that is called chymotrypsinogen which is a single polypetide chain.Since it lacks its 3 side chain characteristics, it is inactive in its action on substrate as it cannot generate Serine alkoxide anion necessary to initiate reaction. This is necessary as an activated chymotrypsin can damage the pancreas itself. On reaching the small intestine Chymotrypsinogen is reacted upon by enzyme called Trypsin., which hydrolyzes Chymotrypisnogen at lysine and arginine and finally activates the Chymotrypsin in small intestine.

e. Activated groups used by proteases are:

Cysteine protease-found in proteinase capthepsin B, Acid protease- in pepsin, Metalloproteases-in carboxypeptidase, etc   

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