Substrate specificity and kinetic properties of pepstatin-insensitive carboxyl proteinase from Pseudomonas sp. No. 101.
The substrate specificity of the pepstatin-insensitive carboxyl proteinase isolated from Pseudomonas sp. No. 101 was studied by using a series of synthetic chromogenic substrates with general structure P5-P4-P3-P2-P1 *(NO2)Phe-Arg-Leu (P5, P4, P3, P2, P1: a variety of amino acids, (NO2)Phe is p-nitro-L-phenylalanine). The nature of the residues occupying the P2, P3 and P4 positions as well as P1 position had strong influences on kinetic parameters. Among those tested, Lys-Pro-Ile-Glu-Phe*(NO2)Phe-Arg-Leu was the best substrate (Km = 3 microM, kcat = 6.9 s-1, kcat/Km = 2300 mM-1 s-1). The S2 subsite of the enzyme was found to contain one or more basic amino acids while the S4 subsite probably includes one or more acidic amino acids. The pH-dependence of the hydrolysis of Ser-Pro-Ala-Lys-Phe*(NO2)Phe-Arg-Leu was studied. The pK1 and pK2 values for enzyme-substrate complex were found to be 2.97 and 4.92, respectively. Coupled with other results, it seems likely that two active carboxyl residues are involved in the catalytic action of the enzyme. In addition, it was found that a specific peptide inhibitor of the enzyme, tyrostatin, is a compeptive inhibitor with a ki value of 2.6 nM.
Oda
K
K
Department of Agricultural Chemistry, Faculty of Agriculture, University of Osaka Prefecture, Sakai, Japan.
Nakatani
H
H
Dunn
B M
BM
D000595:Amino Acid Sequence; D001665:Binding Sites; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D010436:Pepstatins / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D011549:Pseudomonas / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D009842:Oligopeptides; D010436:Pepstatins; D010455:Peptides; D011480:Protease Inhibitors; C014195:Streptomyces pepsin inhibitor; C074130:tyrostatin; D002268:Carboxypeptidases; C031375:pepstatin