Question

Three peptides were obtained from a trypsin digestion of two different polypeptides. Indicate the possible sequences from the given data.

1. Glu-Ala-Phe

2. Val-Val-Arg

3. Val-Met-Lys

Answer 1

Trypsin belongs to the serine protease family and is very similar to chymotrypsin in primary structure. The enzymatic mechanism entails recognition of a target amino acid in a binding pocket and subsequent cleavage of the C-terminal amide bond by a mechanism involving a serine residue on the protease (hence the name). trypsin’s substrate binding pocket is deep and narrower and has a negatively charged aspartate at the bottom of the binding pocket that binds basic amino acids via an ionic interaction. Thus target amino acids for cleavage need to have long side chains and be positively charged to allow formation of the ionic bond. Only arginine and lysine fulfill these criteria that trypsin exclusively cleaves C terminal to arginine or lysine.

If a proline residue is on the carboxyl side of the cleavage site, the cleavage will not occur. If an acidic residue is on either side of the cleavage site, the rate of hydrolysis has been shown to be slower. In other words a generally accepted "Keil rule" is that trypsin cleaves next to arginine or lysine, but not before proline. This represents It cleaves on the carboxyl side of arginine (Arg) and lysine (Lys) residues (but not histidine which is insufficiently basic). We follow N--> C terminii rule for peptides. Hence the peptide sequence will become by Tryptic digestion as follows. 3 peptides but Arg & Lys should not be dead-end = 2 arrangements

1. Val-Val-Arg-Val-Met-Lys-Glu-Ala-Phe           

2. Val-Met-Lys-Val-Val-Arg-Glu-Ala-Phe            

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