Purification and characterization of a new serine proteinase from Bacillus subtilis with specificity for amino acids at P1 and P2 positions.
A proteinase was purified 230-fold to apparent homogeneity from culture filtrates of Bacillus subtilis by a series of column chromatographies on DE52, DEAE-Toyopearl, Cellulofine GC200M, and Mono-Q, using Boc-Ala-Ala-Pro-Ser-pNA as a substrate. The molecular weight of the proteinase was estimated to be 42,000 by SDS-PAGE in the presence of 2-mercaptoethanol. Studies on the substrate specificity with peptide p-nitroanilides and natural peptides revealed that this proteinase preferentially hydrolyzed the peptide bond on the carboxyl-terminal side of either serine or alanine residues at the P1 position and hydrophobic bulky amino acids at P2. It was most active at pH 9.5 for the hydrolysis of Boc-Ala-Ala-Pro-Ser-pNA. The enzyme was inactivated by diisopropyl fluorophosphate (DFP), but not by tosyl-L-phenylalanine chloromethylketone (TPCK) or by EDTA. Based on the reactivity toward substrates and inhibitors, this enzyme differs from elastase- or subtilisin-like proteinase, hence it is a new type of proteinase with specificity for amino acids at P1 and P2 positions.
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka, Japan.
A
A
Yamagata
N
N
Yoshida
K
K
Noda
A
A
Ito
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D007328:Insulin / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D010455:Peptides; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D014862:Wasp Venoms / Q000378:metabolism
D007328:Insulin; D036341:Intercellular Signaling Peptides and Proteins; D009842:Oligopeptides; D010455:Peptides; D015842:Serine Proteinase Inhibitors; D014862:Wasp Venoms; C037500:mastoparan; D012697:Serine Endopeptidases