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

Abstract

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)