Purification and characterization of a thermosensitive X-prolyl dipeptidyl aminopeptidase (dipeptidyl aminopeptidase yscV) from Saccharomyces cerevisiae

Abstract

Dipeptidyl aminopeptidase yscV, a heat-labile enzyme with X-prolyl dipeptidyl aminopeptidase activity, was purified about 470-fold from a protoplast lysate of Saccharomyces cerevisiae. The purification procedure included solubilization of tonoplast-bound activity by the non-ionic detergent octyl-β- d-glucopyranoside, glycerol gradient centrifugation and preparative isoelectric focusing. Sodium dodecyl sulfate polyacrylamide gel electrophoresis resulted in a single band for which a molecular weight of 40 000 was calculated. The peptidase was most active at pH 7.0–7.5 with l-alanyl- l-proline -p- nitroanilide as substrate. Substrate specificity studies indicate that the purified enzyme specifically hydrolyzes peptide bonds involving the carboxyl group of prolyl residues penultimate to unprotected termini unless arginine is the N-terminal amino acid. However, X-Ala-arylamide structures are not attacked. The actinomycete inhibitors antipain, chymostatin and pepstatin had no effect on the enzyme activity, but 5 mM phenylmethylsulfonyl fluoride, an inhibitor of serine peptidases, completely inhibited dipeptidyl aminopeptidase yscV activity. Some heavy metals (Ni 2+, Cd 2+, Zn 2+, Hg 2+) at a concentration of 5·10 −4 M were also found to be potent inhibitors of enzyme activity.