Phaseolain: A PLANT CARBOXYPEPTIDASE OF UNIQUE SPECIFICITY

Abstract

Abstract The mode of action of phaseolain, a carboxypeptidase from French bean leaves, has been studied on a range of proteins and peptides of varying molecular weights. Critical experiments show that phaseolain contains no contaminant aminopeptidase or endopeptidase activity. Kinetic data on the release of COOH-terminal amino acids from polypeptide substrates showed that residues are released in a strictly sequential manner and that phaseolain will release proline and virtually the whole range of amino acids, with the possible exception of aspartic acid. Glutamic acid and glycine are released only slowly. The enzyme unexpectedly released glycinamide from the terminal position of oxytocin but did not release amidated residues from the termini of other peptides tested. The rate of release of COOH-terminal residues from N-benzyloxycarbonyl(Cbz)dipeptide substrates by phaseolain depends in part upon the nature of the COOH-terminal residue, a long chain aliphatic residue being preferred, but more particularly on the nature of the penultimate residue, where an aromatic residue greatly enhances enzyme action. Initial rates of release of leucine from N-Cbz-Phe-Leu are some 400-fold greater than from N-Cbz-Ile-Leu; similarly, proline is readily released from N-Cbz-Tyr-Pro, but is not released from N-Cbz-Gly-Pro under the experimental conditions used. Comparison of kinetic data for two substrates, N-Cbz-Phe-Leu and N-Cbz-Gly-Leu, indicate that the 40-fold difference in initial rate of leucine release by phaseolain (at 0.9 mm substrate) is not so much a consequence of large differences in affinity of enzyme for substrate (Km values are 1.22 ± 0.06 mm and 3.75 ± 0.80 mm, respectively) but is related more to the efficiency of catalysis (Vmax values, 0.82 ± 0.02 and 0.11 ± 0.01, respectively). Conversely, with N-acetyl-Gly-Leu as substrate, alteration of the blocking group hardly affects Vmax values (0.090 ± 0.001 for N-acetyl-Gly-Leu compared with 0.11 ± 0.01 for N-Cbz-Gly-Leu), whereas the lack of affinity of enzyme for the acetylated dipeptide is reflected in a very high Km value of 58.1 ± 0.5 mm. 3-Phenylpropionic acid is a competitive inhibitor of phaseolain (Ki = 1.78 mm with N-Cbz-Phe-Leu as substrate). Although there is general agreement between kinetic data obtained for polypeptide and N-blocked dipeptide substrates, there are differences, notably the very marked inhibitory effect of a penultimate threonine residue on release of leucine (a favorable COOH-terminal residue) from N-Cbz-Thr-Leu, whereas proline (relatively unfavorable COOH-terminal residue) is readily released from the COOH-terminal region of insulin B chain (partial sequence Tyr-Thr-Pro-Lys-Ala).