Characterization of Rarobacter faecitabidus Protease I, a Yeast-Lytic Serine Protease Having Mannose-Binding Activity

Abstract

Rarobacter faecitabidus protease I, a yeast-lytic serine protease, was characterized in order to elucidate the mechanism of lysis of yeast cells by this enzyme. The N-terminal amino acid sequence of the enzyme was found to be homologous to those of Lysobacter enzymogenes alpha-lytic protease and Streptomyces griseus proteases A and B around the catalytic His residue, showing that it is a mammalian type serine protease. In a study of its substrate specificity, it preferentially hydrolyzed the ester of alanine among amino acid p-nitrophenylesters. It also efficiently hydrolyzed succinyl Ala-Pro-Ala p-nitroanilide, the specific synthetic substrate for pancreatic elastase. With oxidized insulin B-chain, it hydrolyzed almost exclusively the peptide bond between valine 18 and cysteic acid 19 in the early step of the reaction, and thereafter it partially hydrolyzed Val12-Glu13, Ala14-Leu15, and Leu15-Tyr16. These results indicate that Rarobacter protease I is elastase-like in its substrate specificity, preferentially hydrolyzing the peptide bond of aliphatic amino acids. Its affinity for yeast cells was also investigated, and while Rarobacter protease I was adsorbed by yeast cells, pancreatic elastase was not. This difference was thought to account for the failure of pancreatic elastase to lyse yeast cells, even though its specificity is similar to that of the yeast-lytic enzyme. Rarobacter protease I was adsorbed by a mannose-agarose column and specifically eluted from the column with a buffer containing D-mannose or D-glucose. These monosaccharides also inhibited its yeast-lytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)