Inhibition of Streptomyces griseus metallo-endopeptidase II (SGMPII) by active-site-directed inhibitors.
Inactivation of Streptomyces griseus metallo-endopeptidase II (SGMPII) by ClCH2CO-DL-(N-OH)Leu-OCH3 and by ClCH2CO-DL-(N-OH)Leu-Ala-Gly-NH2 was studied kinetically. These reagents cause irreversible inhibition of the enzyme in a pseudo-first order reaction, and the inhibition reaction exhibits saturation kinetics. The second-order rate constants for inactivation of SGMPII by ClCH2CO-DL-(N-OH)Leu-OCH3 and by ClCH2CO-DL-(N-OH)Leu-Ala-Gly-NH2 were measured to be 0.12 and 8.9 M-1.s-1, respectively. The order of affinities of metallo-endopeptidases towards these irreversible inhibitors is thermolysin > SGMPII > Pseudomonas aeruginosa elastase. A competitive inhibitor of SGMPII, L-Val-L-Trp, protects the enzyme against inactivation by ClCH2CO-DL-(N-OH)Leu-Ala-Gly-NH2 in a competitive manner. Furthermore, the pH profile of the inactivation closely resembles that for the hydrolysis of synthetic peptide substrates by the enzyme. These findings suggest that these reagents bind reversibly and react irreversibly at the active site of the enzyme.
Kumazaki
T
T
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University.
Ishii
S
S
Yokosawa
H
H
D000595:Amino Acid Sequence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology*; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D013305:Streptomyces griseus / Q000201:enzymology*; D013820:Thermolysin / Q000037:antagonists & inhibitors
D009842:Oligopeptides; C018248:N-chloroacetyl-N-hydroxyleucine methyl ester; C048705:chloroacetyl-N-hydroxyleucyl-alanyl-glycinamide; D010196:Pancreatic Elastase; D008666:Metalloendopeptidases; D013820:Thermolysin; D007930:Leucine