A survey of the kinetic parameters of class C β-lactamases. Cephalosporins and other β-lactam compounds (original) (raw)
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Cefcapene inactivates chromosome-encoded class C β-lactamases
Journal of Infection and Chemotherapy, 2002
The stability of cefcapene and cefpodoxime, oral antibacterial cephalosporins, toward different classes oflactamases was evaluated. For the class A -lactamases, TEM-1, SHV-1, and NMC-A, only the steady-state kinetic parameter (k cat /Km) values were calculated (3100 Ϫ 1.1 ϫ 10 7 M Ϫ1 ·s Ϫ1 ), because these enzymes have very high Km values for cefpodoxime and cefotaxime. As for class B -lactamases L1, IMP-1, and CcrA, in general, similar k cat / Km values were obtained. However, regarding class Clactamases from Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa, and Citrobacter freundii, we found major differences in stability between the two compounds. Cefpodoxime acted as a good substrate for the class C -lactamases, except for the enzyme from E. cloacae; its k cat and Km values were successfully calculated (k cat /Km, 1.8 ϫ 10 5 Ϫ 1.2 ϫ 10 7 M Ϫ1 ·s Ϫ1 ). On the other hand, cefcapene acted as a poor substrate or an inactivator for class Clactamases; its k 2 /K value was successfully calculated (8.7 ϫ 10 5 Ϫ 7.0 ϫ 10 6 M Ϫ1 ·s Ϫ1 ). In addition, k 3 values were determined for -lactamases from P. aeruginosa (2.3 ϫ 10 Ϫ2 ·s Ϫ1 ) and C. freundii (2.1 ϫ 10 Ϫ1 ·s Ϫ1 ). Even though these values could be calculated, transient inactivation as an enzyme reactivation reaction for all these enzymes was observed. These findings suggest the potential of cephem compounds as inhibitors of class C -lactamases.
A survey of the kinetic parameters of class C β-lactamases. Penicillins
Biochemical Journal, 1988
The interaction between six class C beta-lactamases and various penicillins has been studied. All the enzymes behaved in a very uniform manner. Benzylpenicillin exhibited relatively low kcat. values (14-75 s-1) but low values of Km resulted in high catalytic efficiencies [kcat./Km = 10 X 10(6)-75 X 10(6) M-1.s-1]. The kcat. values for ampicillin were 10-100-fold lower. Carbenicillin, oxacillin cloxacillin and methicillin were very poor substrates, exhibiting kcat. values between 1 x 10(-3) and 0.1 s-1. The Km values were correspondingly small. It could safely be hypothesized that, with all the tested substrates, deacylation was rate-limiting, resulting in acyl-enzyme accumulation.
The diversity of the catalytic properties of class A β-lactamases
Biochemical Journal, 1990
The catalytic properties of four class A beta-lactamases were studied with 24 different substrates. They exhibit a wide range of variation. Similarly, the amino acid sequences are also quite different. However, no relationships were found between the sequence similarities and the substrate profiles. Lags and bursts were observed with various compounds containing a large sterically hindered side chain. As a group, the enzymes could be distinguished from the class C beta-lactamases on the basis of the kappa cat. values for several substrates, particularly oxacillin, cloxacillin and carbenicillin. Surprisingly, that distinction was impossible with the kappa cat./Km values, which represent the rates of acylation of the active-site serine residue by the beta-lactam. For several cephalosporin substrates (e.g. cefuroxime and cefotaxime) class A enzymes consistently exhibited higher kappa cat. values than class C enzymes, thus belying the usual distinction between ‘penicillinases’ and ‘ceph...
Biochemistry, 1985
The hydrolysis of cephalosporins containing good leaving groups at the 3'-position [those used in this study were the chromogenic cephalosporin PADAC [pyridine-2-azo-4'-(N',N'-dimethylaniline) substituted on cephalosporin], cephaloridine, and cephalothin], catalyzed by the Staphylococcus aureus PC 1 8-lactamase, proceeds in two spectrophotometrically observable phases. The first involves formation of an acyl-enzyme intermediate while the second involves partitioning of this intermediate between two pathways. One path yields the normal cephalosporoate (3) from which the 3'4eaving group is spontaneously eliminated in solution to give the 3-methylenedihydrothiazine 2, while the second involves initial elimination of the 3' substituent, thus yielding a second acyl-enzyme intermediate, which then hydrolyzes to give the same final product as from the first pathway. The second acyl-enzyme is relatively inert to hydrolysis (t l p N 10 min at 20 "C), and its formation thus leads to transient inhibition of the enzyme. The partition ratio between hydrolysis and elimination at the enzyme active site could be determined either spectrophotometrically from burst experiments or from measurements of residual @-lactamase activity as a function of cephalosporin concentration. This ratio varied with the leaving group ability of the 3' substituent (acetoxy > N,N-dimethylaniline-Caz0-2'-pyridinium > pyridinium) in the anticipated fashion. The inert acyl-enzyme intermediate was isolated by exclusion chromatography and shown to contain the cephem nucleus, but not the 3' substituent, covalently bound to the enzyme. As would be expected, PADAC, cephaloridine, and cephalothin yielded the same inert intermediate. Cephalosporins with poor or no 3'4eaving groups, e.g., dansylcephalothin and desacetoxycephalothin, neither displayed the branched pathway nor yielded the long-lived acyl-enzyme. %e cephalosporins (1) are currently widely used p-lactam CO, H
Biochemical Journal, 1991
The influence of C-6 alpha- or C-7 alpha-methoxylation of the beta-lactam ring in the catalytic action of class A and B beta-lactamases has been investigated. For this purpose the kinetic behaviour of beta-lactamases I (class A) and II (class B) from Bacillus cereus was analysed by using several cephamycins, moxalactam, temocillin and related antibiotics. These compounds behaved as poor substrates for beta-lactamase II, with high Km values and very low catalytic efficiencies. In the case of beta-lactamase I, the substitution of a methoxy group for a H atom at C-7 alpha or C-6 alpha decreased the affinity of the substrates for the enzyme. Furthermore, the acylation of cephamycins was completely blocked, whereas that of penicillins was slowed down by a factor of 10(4)-10(5), acylation being the rate-determining step of the process.
Approaches to the simultaneous inactivation of metallo- and serine-β-lactamases
Bioorganic & Medicinal Chemistry Letters, 2009
A series of cephalosporin-derived reverse hydroxamates and oximes were prepared and evaluated as inhibitors of representative metallo-and serine-β-lactamases. The reverse hydroxamates showed submicromolar inhibition of the GIM-1 metallo-β-lactamase. With respect to interactions with the classes A, C, and D serine β-lactamases, as judged by their correspondingly low Km values, the reverse hydroxamates were recognized in a manner similar to the non-hydroxylated N-H amide side chains of the natural substrates of these enzymes. This indicates that, with respect to recognition in the active site of the serine β-lactamases, the O=C-NR-OH functionality can function as a structural isostere of the O=C-NR-H group, with the NO-H group presumably replacing the amide N-H group as a hydrogen bond donor to the appropriate backbone carbonyl oxygen of the protein. The reverse hydroxamates, however, displayed k cat values up to three orders of magnitude lower than the natural substrates, thus indicating substantial slowing of the hydrolytic action of these serine β-lactamases. Although the degree of inactivation is not yet enough to be clinically useful, these initial results are promising. The substitution of the amide N-H bond by N-OH may represent a useful strategy for the inhibition of other serine hydrolases.
Classification of beta-lactamases: groups 1, 2a, 2b, and 2b
Antimicrobial Agents and Chemotherapy, 1989
Information on 3-lactamases has grown rapidly within the past few years, such that it has become difficult to make meaningful comparisons between well-established P-lactamases and those enzymes recently identified. The intention of this review, therefore, is to present a compilation of data that will be useful in establishing a set of unique characteristics for many of the f3-lactamases that have been described in the primary literature (also see reference 14). The criteria used for characterization are discussed elsewhere (13). PARAMETERS In the accompanying tables, the following designations are used. If an enzyme has been named, both the original name and any later assignments are listed in column 1. When major differences were observed between constitutive enzymes from the same species, all the enzymes are listed. Column 2 indicates whether P-lactamase production is chromosomal or plasmid mediated. The plasmid name is included when appropriate. No entry in this column indicates that the mechanism for production has not been designated. The original host for an enzyme is listed with the appropriate strain number, when available. Substrate profiles were compiled by using the following antibiotics: the penicillins benzylpenicillin, ampicillin, carbenicillin, and cloxacillin; the cephalosporins cephaloridine, cephalothin, cefotaxime, and ceftazidime; the monobactam aztreonam; and the carbapenem imipenem. When possible, Vmax values are listed rather than relative rates of hydroly-TABLE 1. Group 1: cephalosporin-hydrolyzing P-lactamases not inhibited by clavulanic acid (CEP-N)a Produc-Relative rate of hydrolysis Ki for inhibition (,uM) * 0.1 mM substrate for cefotaxime and ceftazidime with the cephalosporinase from P. aeruginosa 10662 (73). q From active site sequence of P. aeruginosa 18 S (45). *Similar to S. marcescens GN7647 cephalosporinase (89). Data for GN7647 cephalosporinase of molecular weight 37,000 (89). 'Also K. Bush, unpublished data.
Interactions of Ceftobiprole with -Lactamases from Molecular Classes A to D
Antimicrobial Agents and Chemotherapy, 2007
The interactions of ceftobiprole with purified β-lactamases from molecular classes A, B, C, and D were determined and compared with those of benzylpenicillin, cephaloridine, cefepime, and ceftazidime. Enzymes were selected from functional groups 1, 2a, 2b, 2be, 2d, 2e, and 3 to represent β-lactamases from organisms within the antibacterial spectrum of ceftobiprole. Ceftobiprole was refractory to hydrolysis by the common staphylococcal PC1 β-lactamase, the class A TEM-1 β-lactamase, and the class C AmpC β-lactamase but was labile to hydrolysis by class B, class D, and class A extended-spectrum β-lactamases. Cefepime and ceftazidime followed similar patterns. In most cases, the hydrolytic stability of a substrate correlated with the MIC for the producing organism. Ceftobiprole and cefepime generally had lower MICs than ceftazidime for AmpC-producing organisms, particularly AmpC-overexpressing Enterobacter cloacae organisms. However, all three cephalosporins were hydrolyzed very slowly...