Novel mechanism of hydrolysis of therapeutic beta-lactams by Stenotrophomonas maltophilia L1 metallo-beta-lactamase.
Stopped-flow tryptophan fluorescence under single turnover and pseudo-first-order conditions has been used to investigate the kinetic mechanism of beta-lactam hydrolysis by the Stenotrophomonas maltophilia L1 metallo-beta-lactamase. For the cephalosporin substrates nitrocefin and cefaclor and the carbapenem meropenem, a substantial quench of fluorescence is observed on association of substrate with enzyme. We have assigned this to a rearrangement event subsequent to formation of an initial collision complex. For the colorimetric compound nitrocefin, decay of this dark inter- mediate represents the overall rate-determining step for the reaction and is equivalent to decay of a previously observed state in which the beta-lactam amide bond has already been cleaved. For both cefaclor and meropenem, the rate-determining step for hydrolysis is loss of a second, less quenched state, in which, however, the beta-lactam amide bond remains intact. We suggest, therefore, that the mechanism of hydrolysis of nitrocefin by binuclear metallo-beta-lactamases may be atypical and that cleavage of the beta-lactam amide bond is the rate-determining step for breakdown of the majority of beta-lactam substrates by the L1 enzyme.
Spencer
J
J
Department of Pathology and Microbiology, University of Bristol School of Medical Sciences, University Walk, Bristol BS8 1TD, United Kingdom. Jim.Spencer@bristol.ac.uk
Clarke
A R
AR
Walsh
T R
TR
D002384:Catalysis; D002433:Cefaclor / Q000378:metabolism; D002511:Cephalosporins / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006868:Hydrolysis; D007202:Indicators and Reagents / Q000494:pharmacology; D007700:Kinetics; D000077731:Meropenem; D008956:Models, Chemical; D013050:Spectrometry, Fluorescence; D020615:Stenotrophomonas maltophilia / Q000201:enzymology*; D013696:Temperature; D013845:Thienamycins / Q000378:metabolism; D013997:Time Factors; D014364:Tryptophan / Q000378:metabolism; D014466:Ultraviolet Rays; D001618:beta-Lactamases / Q000378:metabolism*
D002511:Cephalosporins; D007202:Indicators and Reagents; D013845:Thienamycins; D002433:Cefaclor; D014364:Tryptophan; C088032:beta-lactamase L1; D001618:beta-Lactamases; C021720:nitrocefin; D000077731:Meropenem