Carboxypeptidases from Germinating Soybeans. I. Purification and Properties of Two Carboxypeptidases

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Abstract
Two kinds of carboxypeptidases, CPase Sa and CPase Sb, were separated from germinating soybeans (Glycine max. Akiyoshi) in a highly purified form. The enzymes liberated neutral, acidic, and basic amino acids including proline from the C-termini of N-substituted dipeptides at varying rates. Of the substrates tested, Z [benzyloxycarbonyl]-Gly-Pro, Z-Pro-Pro, Z-Glu-Pro, Bz [benzyl]-Gly-Lys, and Bz-Gly-Arg were hydrolyzed very slowly. The specific activities of purified CPase Sa and Sb were 0.11 and 3.68 units/mg of protein, respectively, for Z-Glu-Phe. The enzyme also released C-terminal amino acid residues from peptide and protein substrates. They possessed weak esterase activity, but were free of endopeptidase and aminopeptidase activities. They had a pH optimum at 5.5 for Z-Glu-Phe. CPase Sb was strongly inhibited by diisopropylfluorophosphate (DFP) and HgCl2, whereas CPase Sa was only partially inhibited. Other metal ions, anions, EDTA, o-phenathroline, N-ethylmaleimide (NEM), p-chloromercuribenzoic acid (PCMB), dithiothreitol (DTT), and monoiodoacetic acid showed no significant effect on the enzymic activity. The MW of CPase Sa was 99,000 (s20,w [sedimentation constant20,w] = 6.9S, D20,w [density20,w] = 6.4 .times. 10-7 cm2/s) and that of CPase Sb, 106,000 (s20,w = 6.1S, D20,w = 5.4 .times. 10-7 cm2/s).

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