Chymotrypsin sequencing peptides with

For example, chymotrypsin cleaves peptides on the C-terminal side of aromatic amino acid residues phenylalanine, tyrosine, and tryptophan, and to a lesser extent some other residues with bulky side-chains, e.g. Leu, Met, Asn, Gin. On the other hand, trypsin cleaves peptides on the C-terminal side of the basic residues arginine and lysine. Elastase usually catalyses hydrolysis of peptide bonds on the C-terminal side of neutral aliphatic amino acids, especially glycine or alanine. These three pancreatic enzymes are about 40% identical in their amino acid sequences, and their catalytic mechanisms are nearly identical. 

From a rare fungus you have isolated an octapeptide that prevents baldness, and you wish to determine the peptide sequence. The amino acid composition is Lys(2), Asp, Tyr, Phe, Gly, Ser, Ala. Reaction of the intact peptide with FDNB yields DNP-alanine plus 2 moles of e-DNP-lysine on acid hydrolysis. Cleavage with trypsin yields peptides the compositions of which are (Lys, Ala, Ser) and (Gly, Phe, Lys), plus a dipeptide. Reaction with chymotrypsin releases free aspartic acid, a tetrapeptide with the composition (Lys, Ser, Phe, Ala), and a tripeptide the composition of which, following acid hydrolysis, is (Gly, Lys, Tyr). What is the sequence ... 

To establish the amino acid sequence unequivocally it is necessary to have peptides with overlapping sequences. This may be accomplished by determining the sequence of fragments obtained from treating a second aliquot of the protein with chymotrypsin. If these fragments are then treated with trypsin as a check, peptides identical to those obtained previously by successive treatment with trypsin and chymotrypsin are obtained. Other proteolytic enzymes, such as pepsin, subtilisin, and papain, with wider specificity than trypsin and chymotrypsin have proved useful in sequencing of some proteins. 

A peptide with 16 amino acid residues is treated with trypsin to give three fragments, while treatment with chymotrypsin gives four fragments (shown). Identify the sequence of the 16 amino acid residues in the starting peptide. 

More specific evidence came from affinity labeling with molecules which could react with specific amino acid group sat or adjacent to the substrate site. These labels were substrate analogues and competitive inhibitors. Substituted aryl alkyl ketones were used. TV-p-toluene-sulphonyl-L-phenylalanine chloromethyl ketone (TPCK) blocked the activity of chymotrypsin. Subsequent sequence analysis identified histidine 57 as its site of binding (see Hess, 1971, p 213, The Enzymes, 3rd ed.). Trypsin, with its preference for basic rather than aromatic residues adjacent to the peptide bond, was not blocked by TPCK but was susceptible to iV-p-toluenesulphonyl-L-lysine chloromethyl ketone (TLCK) (Keil, ibid, p249). 

Ambler and Meadway have very recently reported on their studies of the amino acid sequence of /3-lactamases from representative strains of S. aureus and B. licheniformis (63, 64). The complete sequence of the /3-lactamase protein of S. aureus PCI (type A) was determined by the characterization of the peptides produced by digestion with trypsin, chymotrypsin, or pepsin. 

Chymotrypsin cleaves a peptide selectively at the carboxyl group of amino acids that have aromatic side chains. The side chain of phenylalanine is a benzyl group, C6H5CH2—. If the dipeptide isolated after treatment with chymotrypsin contains valine (V) and phenylalanine (F), its sequence must be VF. 

Fig. 2. The use of overlapping fragments to determine the sequence of a peptide. The protein is first digested with trypsin and the resulting peptides separated and sequenced. The protein is separately digested with chymotrypsin and the resulting peptides again separated and sequenced. The order of the peptide fragments in the protein can be determined by comparing the sequences obtained.

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