No abstract

From the crystal structure of the Bacillus licheniformis 749/C beta-lactamase, energy-minimized structures for the precatalytic, the acyl-enzyme intermediate, and the acylated linear inactivating species for sulbactam--a clinically useful mechanism-based inactivator for class A beta-lactamases--were generated. The effect of individual Ser-235-Ala and Arg244-Ser point mutations on the inactivation and turnover processes was consistent with the existence of hydrogen bonds between the side chains of these residues and the sulbactam species. The departure of the sulfinate leaving group from the acyl-enzyme intermediate of sulbactam is believed to be a prerequisite for the inactivation process. In order to explore the influence of the leaving group, penicillanic acid (2), penicillanic acid alpha-S-oxide (3), and penicillanic acid beta-S-oxide (4) were synthesized and studied in kinetic experiments with the TEM-1 beta-lactamase. Penicillanic acid is only a substrate, but penicillanic acid S-oxides were both substrates and inactivators for the enzyme. An argument is presented to rationalize these observations on the basis of the leaving ability of thiolate, sulfenate, and sulfinate from the acyl-enzyme intermediates of penicillanic acid (2), the penicillanic acid S-oxides (3 and 4), and sulbactam, respectively. The departure of the leaving group does not appear to be rate limiting in the inactivator process, but is an indispensable component of the irreversible inactivation of the enzyme. Molecular dynamics calculations of the putative inactivating species suggest that Lys-73, Lys-234, and Ser-130 are three likely residues that may be modified in the course of the inactivation chemistry. A discussion is presented of the mechanism of formation of the transiently inhibited enzyme species, which comes about as a consequence of the tautomerization of the double bond of the inactivating iminium moiety. In addition, the mechanistic details presented for sulbactam are compared and contrasted with those of clavulanic acid, another clinically used inactivator for class A beta-lactamases.

The role of Ser-235 in the catalytic mechanism of the TEM-1 1-lactamase has been explored by the study of a mutant enzyme in which Ser-235 has been substituted by alanine (Ala-235 mutant enzyme). A comparative kinetic analysis of both the wild-type and the Ala-235 TEM-1 enzymes revealed little effect of this substitution of residue 235 on the turnover of penicillins but a greater effect on the turnover of cephalosporins.Susceptibility testing of Escherichia coli strains harboring the wild-type TEM-1 13-lactamase and the Ala-235 mutant enzyme revealed an effect of the mutation similar to that observed in the enzymological studies. The MICs of two representative cephalosporins for the strain containing the mutant enzyme were much lower than those for the isogenic strain bearing the wild-type TEM-1 13-lactamase. On the other hand, the strain with the mutant enzyme was still highly resistant to penicillins. P-Lactamase activity is the primary mechanism of highlevel bacterial resistance to ,-lactam antibiotics. One strategy to counter ,-lactamase activity has relied on the use of new P-lactam drugs that show more resistance to inactivation by ,3-lactamases (5, 29). Alternatively, the use of inactivators for f-lactamases has extended the clinical utility of ,B-lactamase-susceptible penicillins (25, 27). A structurebased knowledge of the details of how substrates and inactivators bind the active site of a 1-lactamase and are processed by the enzyme is a necessary first step toward understanding the manner in which these enzymes function and evolve. The recent availability of high-resolution crystal structures for class A ,-lactamases (3,11,12,14,20,23,24,28,31) permits such a detailed analysis of the interactions of both substrates and inactivators with the active sites of these enzymes. These investigations will be indispensable for the rational design of future generations of ,-lactam molecules.The interactions of a charged moiety, such as a carboxylate, with the active sites of enzymes are believed to be of high energy and strong (7,33). It was suggested at first that the carboxylate moiety in P-lactam molecules interacts with the side chains of Lys-234 and Ser/Thr-235 of class A 1-lactamases (3,11,12,15,20,23,24,28). Subsequently, we proposed that Arg-244, Ser/Thr-235, and Ser-130, conserved in 3-lactamases, all contribute to the binding of the carboxylate in the process of substrate recognition by these enzymes (36). The recently reported high-resolution crystal structure for the Bacillus lichenifonnis P-lactamase (20) been an evolutionary adaptation by ,B-lactamases to accommodate substrates with different spatial orientations of their carboxylates. For example, the C-3 carboxylate in penicillins is appended to a stereogenic carbon, whereas the C-4 carboxylate in cephalosporins is attached to an sp2-and hence achiral-carbon.A kinetic analysis ofjudiciously designed mutant enzymes provides a powerful tool for exploring the details of the existing models for interactions between small molecules and proteins (4,...

The mutation of Arg-244 to Ser (Arg-244->Ser mutation) in the TEM-1 ,-lactamase has been shown to produce resistance to inactivation by clavulanate in the mutant enzyme and resistance to ampicillin plus clavulanate in a strain of Escherichia coli producing this enzyme. The Arg-164->Ser mutation in the TEM-1 I-lactamase (TEM-12 enzyme) is known to enhance the activity of the enzyme against ceftazidime, resulting in resistance to the drug in a strain producing the mutant enzyme (D. A. Weber, C. C. Sanders, J. S. Bakken, and J. P. Quinn, J. Infect. Dis. 162:460-465, 1990). The doubly mutated derivative of the TEM-1 enzyme (Ser-164/Ser-244) retains the characteristics of the Ser-164 mutant enzyme, i.e., enhanced activity against ceftazidime and sensitivity to inactivation by clavulanate. It also confers the same phenotype as the Ser-164 mutant enzyme, i.e., resistance to ceftazidime and ampicillin, with reversal of this resistance in the presence of clavulanate. Thus, the Arg-164->Ser mutation in the TEM-1 ,-lactamase suppresses the effect of the Arg-244->Ser mutation which, by itself, reduces the sensitivity of the enzyme to inactivation by clavulanate.The production of f-lactamases is the most common bacterial mechanism of resistance to ,-lactam antibiotics. Among clinical isolates of enteric gram-negative bacilli, such as Escherichia coli, the most prevalent plasmid-encoded ,-lactamase is the prototypical class A (1), or group 2b (4, 5), enzyme, TEM-1 (22). This enzyme is produced constitutively and is principally active against penicillins, such as ampicillin, rather than extended-spectrum cephalosporins, monobactams, or carbapenems (5). However, a single mutation, resulting in the replacement of in TEM-1 by serine to produce the TEM-12 enzyme, is sufficient to enhance enzymatic activity against ceftazidime and aztreonam so that clinically significant resistance to these 1-lactams is also conferred (16,28).In an attempt to anticipate the possibility that mutation of the TEM-1 3-lactamase could reduce the inactivation of the enzyme by 3-lactamase inactivators, we mutagenized a laboratory strain of E. coli bearing the TEM-1 3-lactamase with Nmethyl-N'-nitro-N-nitrosoguanidine and grew the mutagenized culture in the presence of inhibitory concentrations of ampicillin and clavulanate (17). In one resistant mutant, the single mutation observed in the structural gene of the enzyme was associated with the replacement of Arg-244 by cysteine.The Cys-244 mutant enzyme and a Ser-244 TEM-1 r-lactamase derivative obtained by site-specific mutagenesis were both resistant to inactivation by clavulanate and conferred resistance to ampicillin and clavulanate (17,18). Some clinical isolates of E. coli resistant to the combination of amoxicillin plus clavulanate have been reported to contain Cys-244 or Ser-244 derivatives of TEM-1 ,B-lactamase (3, 27). The critical role of Arg-244 in the TEM-1 enzyme in the chemistry of inactivation of clavulanate has been elucidated elsewhere (9).In reports of extended-spectrum mutants of TEM-1 ...