| Abstract
| - The mechanism of hydrolysis of cefepime, a novel cephalosporin, bythe class A TEMpUC19 β-lactamasehas been investigated. Models for the active-site binding of thisantibiotic indicate severe steric interactions betweenthe active site of the enzyme and the C7βfunction of cefepime. Specific interactions with the side-chainfunctionsof Pro-167 and Asn-170, amino acids present in the Ω-loop spanningresidues 164−179, have been singled out asimportant in the interactions with the antibiotic. Theseinteractions displace the hydrolytic water (Wat-712) fromitspreferred position for the deacylation step. These observationsare consistent with experimental evidence thatdeacylation is the rate-limiting step in the turnover of cefepime bythis β-lactamase. Furthermore, it has been shownin circular-dichroic measurements that hydrolysis of cefepime by thisβ-lactamase is accompanied by an unprecedentedrelaxation of the structure of the enzyme in order to accommodate thebulky C7β side chain of the antibiotic intheactive site. These findings are in good agreement with dynamicssimulations of the structure of the acyl-enzymeintermediate, which support the possibility for the structuralrelaxation of the protein once this intermediate forms.The class C β-lactamase Q908R, lacking the Ω-loop structuralmotif, turns over cefepime, and the kinetic parametersfor this process were evaluated. In contrast to the class Aβ-lactamase which we studied, the kinetics weresufficientlyfast that circular-dichroic experiments with the Q908R enzyme duringhydrolysis of cefepime could not be carriedout. Two mutant variants of the class A TEMpUC19β-lactamase, Asp-179-Gly and Arg-164-Asn, were preparedtoexplore whether an enlargement of the active site would facilitateturnover of cefepime. Both mutant enzymesshowed improved interactions with cefepime, consistent with ourexpectations. Kinetic analyses for turnover ofcefepime by the parental enzyme and both of its mutant derivatives arepresented.
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