## Doctoral Dissertations

#### Title

The interaction of $\beta$-lactams and $\beta$-lactam binding enzymes: A computational study using molecular modeling, semi-empirical, and {\it ab initio\/} methods

January 1995

#### Keywords

Health Sciences, Pharmacology|Biophysics, Medical

Ph.D.

#### Abstract

Bacterial resistance to $\beta$-lactam antibiotics, such as the penicillins and cephalosporins, has increased due to the evolution and spread of defensive enzymes, the $\beta$-lactamases. The $\beta$-lactamases have been characterized kinetically and structurally as efficient enzymes which hydrolyze $\beta$-lactams at rates near the diffusion limit. The high turnover limits experimental methods and questions about the reaction mechanism persist. Computational methods can be used on this time scale and were applied to evaluate four aspects of $\beta$-lactam/$\beta$-lactamase interactions.^ Class A $\beta$-lactamases have a highly conserved residue, Ser130, whose role in structural integrity and substrate specificity was evaluated. Minimized complexes of substrates and the ROB-1 $\beta$-lactamase were correlated with enzyme kinetic data from site-directed mutagenesis. The importance of a hydroxyl moiety at residue 130 for cephalosporin binding was evident in strong hydrogen bonds to the $\beta$-lactam carboxylate, and the preference of Class A $\beta$-lactamases for penicillins was explained in terms of the hydrogen bonding network.^ Second, the reaction mechanism of clavulanic acid and sulbactam, mechanism based inactivators, was re-evaluated in light of high resolution crystallographic structures. The preacyl and acyl-enzyme clavulanate models indicate Arg244 positions a water (Wat673) for proton transfer in the formation of the acyclic, inactivating species. Molecular dynamics simulations indicate that Lys73 and Ser130 may be the secondary nucleophiles in the inactivation pathway.^ Third, the free energy perturbation method was used to compute a relative binding constant for the core penicillin and cephalosporin structures. New parameters for $\beta$-lactams were developed for AMBER, and a method for perturbation of atoms in rings was refined. The computed value was within 28% of the experimental value, and the hydrophobicity of the penicillin was established as the major component in substrate binding.^ Finally, the capacity of the thiazolidine ring of penicillins to adopt two conformations was studied with the Potential of Mean Force method. The transition barrier between the states was estimated as 3.8 kcal/mol, and the open conformation was determined to be 5.6 kcal/mol more stable in solution. The closed conformation is ejected from the $\beta$-lactamase active site. ^

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