Substrate specificity of pepstatin-insensitive carboxyl proteinase from Bacillus coagulans J-4.
Bacillus coagulans J-4 carboxyl proteinase, designated as J-4, is characterized as alcohol-resistant and insensitive to aspartic proteinase inhibitors such as pepstatin, diazoacetyl-DL-norleucinemetylester, and 1,2-epoxy-3-(p-nitrophenoxy)propane. Here, its substrate specificity was elucidated by using two series of chromogenic substrates, Lys-Pro-Ala-Lys-Phe*Nph (p-nitrophenylalanine:* is cleavage site)-Arg-Leu (XVI) and Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu (RS6), in which the amino acid residues at positions P5-P2, P2', and P3' were systematically substituted. Kinetic parameters were determined for both sets of peptides. J-4 was shown to hydrolyze Lys-Pro-Ala-Ala-Phe-Nph-Arg-Leu most effectively among the XVI series. The kinetic parameters of this peptide were Km = 20.0 +/- 3.24 microM, kcat = 15.4 +/- 0.71 s-1, and kcat/Km = 0.769 +/- 0.128 microM-1.s-1. Among the RS6 series, Lys-Pro-Ile-Pro-Phe-Nph-Arg-Leu was hydrolyzed most effectively. The kinetic parameters of this peptide were Km = 13.7 +/- 1.30 microM, kcat = 9.65 +/- 0.38 s-1, and kcat/Km = 0.704 +/- 0.072 microM-1.s-1. These systematic analyses revealed that J-4 had a unique preference for the P2 position: J-4 preferentially hydrolyzed peptides having an Ala or Pro residue in the P2 position. Other carboxyl proteinases preferred peptides having hydrophobic and bulky amino acid residue such as Leu in the P2 position. Thus, J-4 was found to differ considerably in substrate specificity from the other carboxyl proteinases reported so far.
Shibata
M
M
Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Sakyo-ku, Kyoto, 606-8585, Japan.
Dunn
B M
BM
Oda
K
K
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D002268:Carboxypeptidases / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D010436:Pepstatins / Q000494:pharmacology*; D011487:Protein Conformation; D013379:Substrate Specificity
D010436:Pepstatins; C014195:Streptomyces pepsin inhibitor; D002268:Carboxypeptidases; C031375:pepstatin