The mechanism of hydrolysis of cefepime, a novel cephalosporin, by the class A TEM(pUC19) beta-lactamase has been investigated. Models for the active-site binding of this antibiotic indicate severe steric interactions between the active site of the enzyme and the C-7 beta function of cefepime. Specific interactions with the side-chain functions of Pro-167 and Asn-170, amino acids present in the Omega-loop spanning residues 164-179, have been singled out as important in the interactions with the antibiotic. These interactions displace the hydrolytic water (Wat-712) from its preferred position for the deacylation step. These observations are consistent with experimental evidence that deacylation is the rate-limiting step in the turnover of cefepime by this beta-lactamase. Furthermore, it has been shown in circular-dichroic measurements that hydrolysis of cefepime by this beta-lactamase is accompanied by an unprecedented relaxation of the structure of the enzyme in order to accommodate the bulky C-7 beta side chain of the antibiotic in the active site. These findings are in good agreement with dynamics simulations of the structure of the acyl-enzyme intermediate, which support the possibility for the structural relaxation of the protein once this intermediate forms. The class C beta-lactamase Q908R, lacking the Psi-loop structural motif, turns over cefepime, and the kinetic parameters for this process were evaluated. In contrast to the class A beta-lactamase which we studied, the kinetics were sufficiently fast that circular-dichroic experiments with the Q908R enzyme during hydrolysis of cefepime could not be carried out. Two mutant variants of the class A TEM(pUC19) beta-lactamase, Asp-179-Gly and Arg-164-Asn, were prepared to explore whether an enlargement of the active site would facilitate turnover of cefepime. Both mutant enzymes showed improved interactions with cefepime, consistent with our expectations. Kinetic analyses for turnover of cefepime by the parental enzyme and both of its mutant derivatives are presented.