1. Suppose chymotrypsin is labeled by producing an enzyme that contains a partic

Question

1. Suppose chymotrypsin is labeled by producing an enzyme that contains a particular residue as radioactive. This allows the enzyme to be ‘followed’ throughout experiments and identified easily. Think about the atoms that get ‘labeled’ and the position of these labels throughout each reaction cycle.

A. Suppose the hydrogen of the hydroxyl group of Ser195 is tritiated. 3H-Ser195 chymotrypsin is isolated and its radioactivity confirmed by gel electrophoresis of the recombinant protein and phosphor imaging (phosphor imaging is used to observe radioactive substances on the gel). However, a very frustrating thing occurs after you react your 3H-Ser195 chymotrypsin with peptide – the label disappears! Where did the tritium go? Why is this a poor way to label the enzyme?

Explanation / Answer

1. When a radioactive atom is attached to a molecule, it usually the chemical of this molecule which determines the atom distribution. We can label chymotrypsin by introducing Carbon 14 in place of normal carbon atoms. Carbon 14 or radiocarbon is present n organic compounds and s an important means of carbon dating to find the approximate age of fossils.

Chymotrypsin s a protease and cleaves the carbonyl side of certain peptide bonds by covalent catalysis. t cleaves on the C-terminal phenylalanine tryptophan and tyrosine on peptide chans.  

A] The main substrates of chymotrypsin are peptide bonds n which the amino acid N-terminal to the bond s a tryptophan tyrosine phenylalanine or leucine. Chymotrypsin facilitates the cleavage of a peptide bond by a hydrolysis reaction. Chymotrypsin cleaves peptide bonds by attacking the unreactive carbonyl group with a powerful nucleophile, here, the oxygen atom of a water molecule which has been activated by the basic histidine. The oxygen of water attacks the carbonyl carbon of serine-bound acyl group which, along with other products, releases a proton. Thus, there s a huge chance that the tritium bound hydrogen is the proton which has been released and lost. So, this is not a very reliable way of labeling the enzyme. Introducing carbon 14 is a more efficient way of tracing the enzyme activity.

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