Purification and characterization of the cystinyl bond cleaving yeast aminopeptidase yscXVI

Aminopeptidase yscXVI was purified from the yeast Saccharomyces cerevisiae. By SDS-PAGE the enzyme has a molecular weight of 45,000 Da, and in chromatofocusing, elution was observed at pH 6.2. The synthetic substrate cystinyl-4-nitroanilide (Km 22.5 microM, Vmax 12.9 mU/mg) is cleaved most efficiently in the pH range 7-8. Besides cleaving this standard substrate, aminopeptidase yscXVI acts on several other 4-nitroanilide substrates with unsubstituted N-terminal L-amino acids. Highest hydrolysis rate was measured with Lys-4-nitroanilide and Leu-4-nitroanilide. The activity of aminopeptidase yscXVI is abolished by chelating agents and restored by Zn2+, Mn2+ and Co2+ ions. Bestatin and amastatin are both strong inhibitors of the enzyme, with Ki values of 0.53 microM and 0.93 microM, respectively. Aminopeptidase yscXVI is detectable in the logarithmic growth phase, stationary phase, and in starved cultures of yeast.