Abstract
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.
Highlights
Zinc-dependent or metallo--lactamases [1] are bacterial enzymes of considerable clinical and mechanistic interest
Our results confirm the presence of the previously reported intermediate in nitrocefin hydrolysis, confirm that the rate-determining step in this process occurs after C–N bond cleavage, and show that tryptophan fluorescence of L1 is a suitable probe for investigating conformational changes of the enzyme during turnover of substrates
We initially investigated the utility of tryptophan fluorescence as a probe for events during hydrolysis of the synthetic cephalosporin nitrocefin by the L1 enzyme
Summary
S. maltophilia L1 was expressed and purified as previously described [15, 29]. Nitrocefin was purchased from Becton Dickinson. Steady-state experiments were performed on a PerkinElmer Life Sciences Lamda 2 UV-VIS spectrophotometer equipped with a thermostatted cell holder and circulating water bath to maintain temperature at 10 °C. Stopped-flow experiments were performed on an Applied Photophysics SX18MV apparatus equipped with a constant temperature circulating water bath. Fluorescence data were collected using an excitation wavelength of 295 nm and a WG320 nm cut-on filter on the emission photomultiplier. The photomultiplier input was adjusted for each protein concentration to maintain a total signal change of 1V between protein in the absence of substrate and dark current readings. Data were recorded as absorbance units, with photomultiplier input first adjusted to give a reading of 0 for buffer only at each wavelength used. Residue numbers referred to under “Discussion” are given according to the proposed standard numbering scheme for metallo-lactamases [32]
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