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 enolp yruvoyl transferase [Kahan, F. M., et al. (1974) Ann. N.Y. Acad. Sci. 235, 364], the enzyme responsible for catalyzing the first committed s tep in bacterial cell wall biosynthesis. Time-dependent inactivation o f MurZ by fosfomycin was found to be greatly accelerated by the presen ce of cosubstrate UDP-GlcNAc but could also be speeded appreciably by the unreactive substrate analog 3-deoxy-UDP-GlcNAc. These results argu e against a reaction-based participation of the cosubstrate and sugges t that UDP-GlcNAc has a role in influencing active site conformation c ritical to the inactivation event. A study of the influence of UDP-Glc NAc and fosfomycin on the kinetics of inactivation allowed the determi nation of dissociation constants for fosfomycin (K-F = 8.6 mu M) and U DP-GlcNAc (K-S = 14 mu M), in addition to a limiting inactivation rate constant (k(inact) = 7.4 min(-1)) at saturating UDP-GlcNAc and fosfom ycin concentrations. Mass spectrometry of inactivated MurZ demonstrate d 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 inhibit or per active site when assessed using either enzyme activity or mass spectrometry as an index of modification. Peptide mapping of tryptic d igests of fosfomycin-inactivated MurZ revealed modification of a uniqu e 41-mer, the sequence of which revealed that Cys115 was the site of a ttachment of fosfomycin.