Effect of the 3′-leaving group on turnover of cephem antibiotics by a class C β-lactamase

Abstract

It has been previously demonstrated for class A beta-lactamases and the DD-peptidase of Streptomyces R61 that the presence of a leaving group at the 3'-position of a cephalosporin can lead to the generation of more-inert acyl-enzyme intermediates than from cephalosporins lacking such a leaving group, and thus to beta-lactamase inhibitors and potentially better antibiotics. In the present work we extend this result to a class C beta-lactamase, that of Enterobacter cloacae P99. The effect is not seen with first-generation cephalosporins, since here deacylation generally seems faster than elimination of the leaving group, but it does clearly appear with cephamycins and third-generation cephalosporins. The structural and/or mechanistic features of the active site giving rise to this phenomenon may thus be common to all serine beta-lactamases and transpeptidases.

A rapid‐kinetic study of the class C β‐lactamase of Enterobacter cloacae 908R

“…The inactivation of the enzyme monitored with nitrocefin as a reporter substrate was biphasic with several cephalosporins (cefuroxime, eeftriaxone and ceftazidime) with which a rearrangement of the eephalosporoyl moiety of*.he acyl-enzyme could be susl~cted involving the elimination of the C3' leaving group as proposed by Mazzella and Pratt [10]. The similar behaviour observed with some penicillins might be related with a conformational change such as that described by Citri et al [11].…”

“…2) but could be fitted to the equation: (-k,,t) + B exp (-k,t) where v, and x ,+ are the rates of nitrocefin hydrolysis at time t and at the steady.state respectively (Table IlL This latter behaviour could be explained on the basis of Scheme 1 assuming a slow formation of the HenriMichaelis complex ES. Alternatively, the presence of a potential ieavin~ group on C'.~ of the three cephaIosporins suggested the possibility of the branched pathway depicted by Scheme 1I [10] where ES** represents a second aeyl-enzyme formed by the rearrangement of the cephalosporoyl moiety and P' the product formed by hydrolysis of '.his latter acyl--enzyme. Table 1 were derived by fitting, the r~ults to the equations o1" a h)'perbola (a) or a straisht line (b}.…”

Amino Acid Sequence Determinants of Extended Spectrum Cephalosporin Hydrolysis by the Class C P99 β-Lactamase

“…This value has been shown to correlate strongly with MIC values, and therefore, the higher k cat /K m value is consistent with the higher ceftazidime resistance of the mutant (53). The deacylation process is rate-limiting for hydrolysis of oxyimino ␤-lactams such as ceftazidime by class C ␤-lactamases (51,54). Hence, the large increase in k cat may reflect an increase in the rate of deacylation.…”

“…Because of the mechanism of class C ␤-lactamases, however, an increase in K m does not necessarily indicate less efficient substrate binding. When deacylation is the rate-limiting step, an increased rate of deacylation will also increase the value of K m (54).…”

Mechanistic Basis for the Action of New Cephalosporin Antibiotics Effective against Methicillin- and Vancomycin-resistant Staphylococcus aureus

“…Parenthetically we state here that since the change in the PBP 2a CD spectrum is also seen with penicillins (6), the effect that we observe with cephalosporins 1-3 cannot be attributed to the well characterized tautomerization of the dihydrothiazine double bond of the cephalosporins upon acylation of the enzyme (32,33). Furthermore, the chromophore for the cephalosporins is weak, compared with the signals from the protein, such that it does not influence any of the events seen in Fig.…”