Enzymic determination of C-terminal sequences

A limited amount of information can be obtained by the use of proteolytic enzymes that detach either amino acids or dipeptides sequentially from the C-terminus. They are thus complementary to the aminopeptidases and dipeptidyl aminopeptidases. Two pancreatic enzymes, carboxypeptidases A and B, differ in specificity. The former preferentially liberates C-terminal amino acids with aromatic side chains, somewhat less readily amino acids with alkyl side chains and, more slowly still, other amino acids, but not Pro, Arg, Lys and His. In contrast, carboxypeptidase B releases only C-terminal Arg, Lys and His. Carboxypeptidase Y is much less specific and is capable of removing all amino acids, although Gly and Pro are liberated only slowly. As with aminopeptidases, it is advisable to analyse the hydrolysate at intervals in order to determine the C-terminal sequence of amino acids. An interesting recent development (Carles et al., 1988) uses carboxypeptidase to effect transpeptidation between the protein being sequenced and a tritiated amino acid. The labelled protein is then degraded by various specific methods and then the labelled fragments are isolated by gel electrophoresis and subjected to Edman degradation. 

Dipeptidyl carboxypeptidases remove the C-terminal dipeptide intact and therefore are analogous to the dipeptidyl aminopeptidases such as cathepsin C. One such enzyme, angiotensin-converting enzyme, is important biologically for converting angiotensin I into the hypertensive angiotensin II (see Section 9.3). This enzyme does not hydrolyse bonds of the type X—Pro but will hydrolyse Pro—X bonds. The use of dipeptidyl carboxypeptidases for sequence determination would probably increase if pure enzymes were readily available commercially. 

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