314 Winder and Walkerhydroxyproline, or lysine residues (1). The specificity of carboxypep-
tidase B is far more restricted, cleaving only C-terminal arginine and
lysine residues. Carboxypeptidases A and B, therefore, tended to be
used together, but even so, exopeptidase activity was effectively blocked
when a proline residue was reached. The isolation of carboxypepti-
dase C provided an enzyme that combined the specificity of carboxy-
peptidases A and B, but also cleaved at Pro residues (2), i.e., it cleaves
all C-terminal amino acids. Carboxypeptidase Y (CPY, isolated from
baker's yeast) has the same broad specificity as carboxypeptidase C,
but because of its strong action on protein substrates and its ability to
work in the presence of urea and detergents, it is nowadays the
enzyme of choice for C-terminal sequence analysis and will be
described in this chapter.
- The Enzyme
2.1. Purification
Large-scale purification of the enzyme from baker's yeast has been
described by a number of workers (3,4).
2.2. Specificity
The enzyme cleaves all L-amino acids one residue at a time from the
C-terminal of polypeptide chains. However, the rate of release of indi-
vidual amino acids varies. Catalysis is maximum when the penulti-
mate and/or terminal residues have aromatic or aliphatic side chains
(5). When glycine or aspartic acid is in the terminal position, or lysine
and arginine in the penultimate position, the release of the amino acid
is slow (3,6). The cleavage of tripeptides is difficult, and dipeptides
are completely resistant to hydrolysis. C-terminal proline is a good
substrate, but a proline residue on the carboxy terminal side of glycine
is not likely to be released (5).
The enzyme is a serine carboxypeptidase having a strongly nucleo-
philic serine residue at the active center (7, 8), which is generated by
a charge relay system involving a histidine residue (9). It has strong
esterase activity toward the substrates of chymotrypsin and also
anilidase activity (10). It therefore seems to be quite similar to chy-
motrypsin in both mechanism and active site, although carboxypepti-
dase Y is an exopeptidase and chymotrypsin an endopeptidase.