Kinetics, stoichiometry, and identification of the reactive thiolate in the inactivation of UDP-GlcNAc enolpyruvoyl transferase by the antibiotic fosfomycin.

Fosfomycin [(1R,2S)-1,2-epoxypropylphosphonic acid] has been shown to exert its antibiotic effect through the inhibition of UDP-GlcNAc enolpyruvoyl transferase [Kahan, F. M., et al. (1974) Ann. N.Y. Acad. Sci. 235, 364], the enzyme responsible for catalyzing the first committed step in bacterial cell wall biosynthesis. Time-dependent inactivation of MurZ by fosfomycin was found to be greatly accelerated by the presence of cosubstrate UDP-GlcNAc but could also be speeded appreciably by the unreactive substrate analog 3-deoxy-UDP-GlcNAc. These results argue against a reaction-based participation of the cosubstrate and suggest that UDP-GlcNAc has a role in influencing active site conformation critical to the inactivation event. A study of the influence of UDP-GlcNAc and fosfomycin on the kinetics of inactivation allowed the determination of dissociation constants for fosfomycin (KF = 8.6 microM) and UDP-GlcNAc (KS = 14 microM), in addition to a limiting inactivation rate constant (k(inact) = 7.4 min-1) at saturating UDP-GlcNAc and fosfomycin concentrations. Mass spectrometry of inactivated MurZ demonstrated an increase in molecular weight of 138, consistent with the covalent addition of a molar equivalent of fosfomycin (136 kDa). Titration of MurZ with fosfomycin revealed a stoichiometry of 1 molecule of inhibitor per active site when assessed using either enzyme activity or mass spectrometry as an index of modification. Peptide mapping of tryptic digests of fosfomycin-inactivated MurZ revealed modification of a unique 41-mer, the sequence of which revealed that Cys115 was the site of attachment of fosfomycin.