EVIDENCE FOR STRUCTURAL ELASTICITY OF CLASS-A BETA-LACTAMASES IN THE COURSE OF CATALYTIC TURNOVER OF THE NOVEL CEPHALOSPORIN CEFEPIME

The mechanism of hydrolysis of cefepime, a novel cephalosporin, by the class A TEM(pUC19) beta-lactamase has been investigated. Models for t he active-site binding of this antibiotic indicate severe steric inter actions between the active site of the enzyme and the C-7 beta functio n of cefepime. Specific interactions with the side-chain functions of Pro-167 and Asn-170, amino acids present in the Omega-loop spanning re sidues 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 deacylati on is the rate-limiting step in the turnover of cefepime by this beta- lactamase. Furthermore, it has been shown in circular-dichroic measure ments that hydrolysis of cefepime by this beta-lactamase is accompanie d by an unprecedented relaxation of the structure of the enzyme in ord er to accommodate the bulky C-7 beta side chain of the antibiotic in t he active site. These findings are in good agreement with dynamics sim ulations of the structure of the acyl-enzyme intermediate, which suppo rt the possibility for the structural relaxation of the protein once t his intermediate forms. The class C beta-lactamase Q908R, lacking the Psi-loop structural motif, turns over cefepime, and the kinetic parame ters for this process were evaluated. In contrast to the class A beta- lactamase which we studied, the kinetics were sufficiently fast that c ircular-dichroic experiments with the Q908R enzyme during hydrolysis o f 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 prepare d to explore whether an enlargement of the active site would facilitat e turnover of cefepime. Both mutant enzymes showed improved interactio ns with cefepime, consistent with our expectations. Kinetic analyses f or turnover of cefepime by the parental enzyme and both of its mutant derivatives are presented.