title: Mechanism of inhibition of chromosomal beta-lactamases by third-generation cephalosporins.
abstract: The kinetic interactions of the beta-lactamase from Enterobacter cloacae 908 R with ceftriaxone, cefotaxime, and ceftazidime have been examined in detail. With all of these cephalosporins, there is an initial rapid reaction involving opening of the beta-lactam that then decreases to a slower steady-state rate (kss) of beta-lactam hydrolysis (at 37 degrees C: ceftriaxone, kss = 0.044 s-1; cefotaxime, kss = 0.033 s-1; ceftazidime, kss = 0.011 s-1). More than stoichiometric quantities of beta-lactam are cleaved during the rapid phase, during which there is accumulation of a transiently stable cephalosporin-enzyme complex whose rate of breakdown is slower than the overall rate of hydrolysis. Qualitatively similar behavior is observed with the E. cloacae M6300 beta-lactamase. These observations eliminate the possibility that the reaction follows a simple linear kinetic scheme. A branched kinetic scheme in which an initially formed acyl intermediate partitions between deacylation and elimination of the 3' substituent is proposed to explain the data. Investigations of the interaction of ceftriaxone with the chromosomally encoded beta-lactamases from Citrobacter freundii, Providencia rettgeri, Morganella morganii, Pseudomonas aeruginosa, and Escherichia coli show that the partitioning behavior of E. cloacae beta-lactamases is atypical. All of the data, however, clearly demonstrate that it is a physical impossibility for cephalosporin trapping to contribute to bacterial resistance phenotypes.
mesh_terms: D002439:Cefotaxime / Q000378:metabolism / Q000494:pharmacology; D002442:Ceftazidime / Q000378:metabolism / Q000494:pharmacology; D015296:Ceftizoxime / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D002443:Ceftriaxone / Q000378:metabolism / Q000494:pharmacology; D002511:Cephalosporins / Q000378:metabolism / Q000494:pharmacology*; D055598:Chemical Phenomena; D002621:Chemistry; D002876:Chromosomes, Bacterial; D002954:Citrobacter / Q000201:enzymology / Q000235:genetics; D004754:Enterobacter / Q000201:enzymology / Q000235:genetics; D004755:Enterobacteriaceae / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D011511:Proteus / Q000201:enzymology / Q000235:genetics; D011532:Providencia / Q000201:enzymology / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
chemical_list: D002511:Cephalosporins; D065093:beta-Lactamase Inhibitors; C047444:cefetamet; D002443:Ceftriaxone; D002442:Ceftazidime; D015296:Ceftizoxime; D001618:beta-Lactamases; D002439:Cefotaxime