Novel Mechanism of Hydrolysis of Therapeutic β-Lactams byStenotrophomonas maltophilia L1 Metallo-β-lactamase

Abstract

Stopped-flow tryptophan fluorescence under single turnover and pseudo-first-order conditions has been used to investigate the kinetic mechanism of ␤-lactam hydrolysis by the Stenotrophomonas maltophilia L1 metallo-␤-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 intermediate represents the overall rate-determining step for the reaction and is equivalent to decay of a previously observed state in which the ␤-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 ␤-lactam amide bond remains intact. We suggest, therefore, that the mechanism of hydrolysis of nitrocefin by binuclear metallo-␤-lactamases may be atypical and that cleavage of the ␤-lactam amide bond is the rate-determining step for breakdown of the majority of ␤-lactam substrates by the L1 enzyme.Zinc-dependent or metallo-␤-lactamases (1) are bacterial enzymes of considerable clinical and mechanistic interest. In the clinical context, their ability to hydrolyze the newer generation of carbapenem antibiotics (2), together with the potential for interspecies transfer arising from their presence on mobile genetic elements (3, 4), is a cause for increasing concern as carbapenem use becomes more widespread. The group includes both nonspecific enzymes (able to efficiently hydrolyze penicillin and cephalosporin antibiotics in addition to carbapenems) and specific enzymes (5) and may utilize one or two zinc ions for maximal activity (6 -8). Although mechanistic analogies with zinc peptidases have been attempted (7), it is now clear that metallo-␤-lactamases are members of a distinct superfamily of hydrolases that includes bacterial arylsulfatases and type II glyoxalases (8, 9). Despite increasing attention in recent years (10 -12), understanding of metallo-␤-lactamase mechanism remains incomplete, and there are to date no clinically effective inhibitors.There is general agreement that metallo-␤-lactamases hydrolyze substrate by a nucleophilic attack of zinc-bound water upon the carbonyl carbon of the scissile bond (Ref. 13 and references therein). This would be expected to lead to formation of a tetrahedral oxyanion intermediate that would decay to product via cleavage of the ␤-lactam amide bond and protonation of the amide nitrogen. Several models of such a reaction scheme have been postulated (7,10,14,15); these vary primarily in the source of the proton required for product formation. Experimental evidence points to different conclusions for different enzymes: in the BCII enzyme from Bacillus cereus, a conserved aspartate in the active site appears to shuttle a proton from the oxyanion to the amide nitrogen, and the major intermedia...

Metal Binding Asp-120 in Metallo-β-lactamase L1 from Stenotrophomonas maltophilia Plays a Crucial Role in Catalysis

“…On average, D120N showed ten times less activity toward penicillins than 1 The abbreviations used are: ICP-AES, inductively coupled plasmaatomic emission spectroscopy; L1, metallo-␤-lactamase from S. maltophilia; MTCN, MES-TRIS-CHES-NaCl buffer; CD, circular dichroism; MES, 4-morpholineethanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid. To probe further the binding of nitrocefin to wild-type L1 and the mutants, stopped-flow fluorescence studies were conducted as previously described (52). The reaction of enzyme with nitrocefin under steady-state conditions at 10°C resulted in a rapid decrease in fluorescence followed by a rate-limiting return of fluorescence (data not shown).…”

“…The reaction of enzyme with nitrocefin under steady-state conditions at 10°C resulted in a rapid decrease in fluorescence followed by a rate-limiting return of fluorescence (data not shown). Fitting of the data, as described by Spencer et al (52), yielded K S values for wild-type L1 and the three mutants (Table I).…”

Probing the Dynamics of a Mobile Loop above the Active Site of L1, a Metallo-β-lactamase from Stenotrophomonas maltophilia, via Site-directed Mutagenesis and Stopped-flow Fluorescence Spectroscopy

“…To observe fluorescence changes upon the binding of substrate to wild-type L1 and the mutants, stopped-flow fluorescence studies were conducted as previously described (43). The reaction of enzyme with nitrocefin under single-turnover conditions at 25°C resulted in a rapid decrease in fluorescence followed by a rate-limiting return of fluorescence for all of the single point mutants except W39F (Fig.…”

“…First, each enzyme was analyzed with stoppedflow fluorescence using nitrocefin as the substrate (43,52). W53F, W204F, W206F, and W269F all exhibited fluorescence spectra similar in shape to that of wild-type L1 (Fig.…”

“…1). Spencer and coworkers (43) had demonstrated previously that the quenching of fluorescence and subsequent return to original values in wild-type L1 could be used to monitor substrate binding and catalysis (43). It was, therefore, necessary to determine whether these fluorescence changes could be pinpointed to a single Trp residue.…”