Determination of the kinetic parameters of Escherichia coli leader peptidase activity using a continuous assay: the pH dependence and time-dependent inhibition by beta-lactams are consistent with a novel serine protease mechanism.
Bacterial leader peptidase (LPase) is a potential target for the development of novel anti-infective agents, but to data only peptides based upon natural macromolecular substrates have been reported as inhibitors. In this work is described a continuous assay for Escherichia coli LPase activity, based upon Ac-WSASALAKI-AMC (I) as the substrate, that can be monitored either spectrophotometrically or spectrofluorometrically. The LPase reaction is coupled to the liberation of AMC (aminomethylcoumarin) via a nonspecific leucine aminopeptidase. LPase and a short form of the enzyme (LPase-sf) lacking the membrane spanning domains displayed saturable kinetics toward I. The second-order rate constants were approximately 2 x 10(5) M-1 h-1 at pH 7.5 and were comparable to those reported in the literature for peptide substrates based upon natural cleavage sites in preproteins. LPase was inhibited by beta-lactams. [S-(R*,S*)]-4-[(1-(((1-(5-toluoyl)butyl)amino)carbonyl)-3,3-dimethyl-4- oxo-2-azetidinyl)oxyl]benzoic acid (L-684,-248, 588 microM) inhibited the LPase-catalyzed hydrolysis of 50 microM I and 125 microM Ac-WLVP-Nleu-LSFAAEGDDPA-NH2 by 30% and 88% over 1 and 4 h, respectively. The inhibition of LPase by L-684,248 and its C-4 diasteromer was time dependent and yielded second-order rate constants (kinact/Ki) of 12 and 7.7 M-1 min-1, respectively. The process was structurally specific as the C-3 diethyl substituted beta-lactam (C-4 S-isomer) was inactive. The latter data correlate with the LPase preference for alanine at the P1 position of peptide substrates [Kuo et al. (1993) Arch. Biochem. Biophys. 303, 274-280].(ABSTRACT TRUNCATED AT 250 WORDS)
Kuo
D
D
Department of Enzymology, Merck Research Laboratories, Rahway, New Jersey 07065.
Weidner
J
J
Griffin
P
P
Shah
S K
SK
Knight
W B
WB
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001384:Azetidines / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D021382:Protein Sorting Signals / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D047090:beta-Lactams
D000900:Anti-Bacterial Agents; D001384:Azetidines; D008565:Membrane Proteins; D011480:Protease Inhibitors; D021382:Protein Sorting Signals; D047090:beta-Lactams; C088411:L 684248; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase