title: Purification and characterization of a novel metalloendopeptidase from Saccharomyces cerevisiae.
abstract: We previously identified an activity in the soluble fraction of the yeast Saccharomyces cerevisiae that is a candidate for catalyzing the proteolytic maturation of farnesylated-CXXX precursor polypeptides. We describe here a 1259-fold purification of this activity by chromatography on DEAE-cellulose, hydroxylapatite, phenyl-Sepharose, and Sephacryl S-200. Sodium dodecyl sulfate gel electrophoresis of this preparation demonstrated a single 68-kDa polypeptide chain. The experimentally determined N-terminal amino acid sequence was identical at all 20 positions with residues 28-47 of the deduced sequence of the S. cerevisiae YCL57w gene product. This analysis suggests that the YCL57w gene encodes this enzyme and that the initial translation product may contain a leader peptide. Its complete deduced amino acid sequence shows significant homology to a number of zinc metallopeptidases and is most closely related to rat metalloendopeptidase 24.15 (E.C. 3.4.24.15), an enzyme that preferentially cleaves after hydrophobic residues. Using the purified yeast enzyme, we show a unique cleavage site in the peptides bradykinin and beta-neoendorphin four residues from the C-terminus on the carboxyl side of a hydrophobic amino acid. The cleavage pattern for neurotensin revealed a major site three residues from the C-terminus also on the carboxyl side of a hydrophobic residue and a minor site four residues from the C-terminus of the peptide. This specificity is similar to that of rat endopeptidase 24.15 and may explain why the farnesylated peptide employed in our studies is a good substrate for the yeast enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles 90024-1569.
authors.forename: C A
authors.initials: CA
authors.lastname: Hrycyna
authors.forename: S
authors.initials: S
authors.lastname: Clarke
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011480:Protease Inhibitors / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D010446:Peptide Fragments; D011480:Protease Inhibitors; D017886:Durapatite; D008666:Metalloendopeptidases