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.
Marquardt
J L
JL
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Brown
E D
ED
Lane
W S
WS
Haley
T M
TM
Ichikawa
Y
Y
Wong
C H
CH
Walsh
C T
CT
D019883:Alkyl and Aryl Transferases; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli; D005578:Fosfomycin / Q000737:chemistry*; D007700:Kinetics; D013058:Mass Spectrometry; D010449:Peptide Mapping; D014166:Transferases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000737:chemistry
D001426:Bacterial Proteins; D005578:Fosfomycin; D014537:Uridine Diphosphate N-Acetylglucosamine; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C022566:UDP-N-acetylglucosamine 1-carboxyvinyltransferase; D003545:Cysteine