Kinetics and Mechanism of Inhibition of a Serine β-Lactamase by O-Aryloxycarbonyl Hydroxamates

Abstract

The class C serine β-lactamase of Enterobacter cloacae P99 is irreversibly inhibited by Oaryloxycarbonyl hydroxamates. A series of these new inhibitors has been prepared to investigate the kinetics and mechanism of the inactivation reaction. A pH-rate profile for the reaction indicated that the reactive form of the inhibitor is neutral rather than anionic. The reaction rate is enhanced by electron-withdrawing aryloxy substituents and by hydrophobic substitution on both aryloxy and hydroxamate groups. Kinetics studies show that the rates of loss of the two possible leaving groups, aryloxide and hydroxamate are essentially the same as the rate of enzyme inactivation. Nucleophilic trapping experiments prove, however, that the aryloxide is the first to leave. It is likely, therefore, that the rate-determining step of inactivation is the initial acylation reaction, most likely of the active site serine, yielding a hydroxamoyl-enzyme intermediate. This then partitions between hydrolysis and aminolysis by Lys 315, the latter to form an inactive, cross-linked active site. A previously described crystal structure of the inactivated enzyme shows a carbamate cross-link of Ser 64 and Lys 315. Structure-activity studies of the reported compounds suggest that they do not react at the enzyme active site in the same way as normal substrates. In particular, it appears that the initial acylation by these compounds does not involve the oxyanion hole, an unprecedented departure from known and presumed reactivity. Molecular modeling suggests that an alternative oxyanion hole may have been recruited, consisting of the side chain functional groups of Tyr 150 and Lys 315. Such an alternative mode of reaction may lead to the design of novel inhibitors.For decades now, β-lactams have been one of our most effective weapons against bacterial infections (1). These drugs, although still the first line of attack in many clinical situations, have been compromised to a considerable degree by bacterial resistance to them (2). Among various sources of resistance that have arisen in bacteria, the most generally troublesome is the production of β-lactamases. These enzymes very effectively catalyze the hydrolysis and thus destruction of β-lactams before they can reach their cellular targets (3).The threat posed by β-lactamases to the efficacy of β-lactam antibiotics has been tackled by pharmaceutical companies in several ways. One approach that has been quite successful to date is that of including a β-lactamase inhibitor with a β-lactam antibiotic in combination therapies. For many years now, such combinations, using the now-classical β-lactamase inhibitors clavulanic acid, sulbactam and tazobactam, have been used to advantage (4). Since these inhibitors are themselves β-lactams, however, it is perhaps not surprising to find that certain β-lactamase mutants are capable of hydrolyzing them quite effectively. Such mutants have †

Three Decades of β-Lactamase Inhibitors

“…The proposed inhibition mechanism involves rate-limiting acylation of the hydroxamate. However, the inhibitor is stabilized not by the traditional oxyanion hole comprised of the backbone nitrogens of Ser64 and Ser318 but rather by the side chains of Tyr150 and Lys315 (330). As with clavulanate and tazobactam, the acyl-enzyme proceeds to either hydrolysis or inactivation.…”

“…12) (PDB 2P9V) (450). TEM-2 and OXA-1 ␤-lactamases were also inhibited by O-aryloxycarbonyl hydroxamates, and the MBL GIM-1 was inhibited by a "reverse" hydroxamate (hydroxylamino replacing hydroxylamine) conjugated to a cephalosporin nucleus (131,330). Additional mechanistic studies of hydroxamates and their derivatives, as well as in vitro activity data, will be of much interest.…”

Specificity and mechanism of mandelamide hydrolase catalysis

“…On cooling of the reaction mixture, pure crystals of 4 precipitated. Compound 16 was previously prepared in this laboratory [11,12]. The carbamates 16 and 19 were obtained by reaction of the corresponding hydroxamic acids with trimethylsilylisocyanate (Acros) and 6 purified by recrystallization from aqueous ethanol.…”

“…Molecular modeling simulations were performed on a SGI workstation running the program Insight II, essentially as previously described [12]. The homology structure of MAH [9] was modified to obtain the adduct 14 with the active site Ser 204.…”

Serendipitous Discovery of α-Hydroxyalkyl Esters as β-Lactamase Substrates

“…These molecules were found to be effective against all serine β-lactamases, although particularly so against representative class C enzymes (4, 5). …”