A structure-based analysis of the inhibition of class A beta-lactamases by sulbactam.

From the crystal structure of the Bacillus licheniformis 749/C beta-lactamase, energy-minimized structures for the precatalytic, the acyl-enzyme intermediate, and the acylated linear inactivating species for sulbactam--a clinically useful mechanism-based inactivator for class A beta-lactamases--were generated. The effect of individual Ser-235-Ala and Arg244-Ser point mutations on the inactivation and turnover processes was consistent with the existence of hydrogen bonds between the side chains of these residues and the sulbactam species. The departure of the sulfinate leaving group from the acyl-enzyme intermediate of sulbactam is believed to be a prerequisite for the inactivation process. In order to explore the influence of the leaving group, penicillanic acid (2), penicillanic acid alpha-S-oxide (3), and penicillanic acid beta-S-oxide (4) were synthesized and studied in kinetic experiments with the TEM-1 beta-lactamase. Penicillanic acid is only a substrate, but penicillanic acid S-oxides were both substrates and inactivators for the enzyme. An argument is presented to rationalize these observations on the basis of the leaving ability of thiolate, sulfenate, and sulfinate from the acyl-enzyme intermediates of penicillanic acid (2), the penicillanic acid S-oxides (3 and 4), and sulbactam, respectively. The departure of the leaving group does not appear to be rate limiting in the inactivator process, but is an indispensable component of the irreversible inactivation of the enzyme. Molecular dynamics calculations of the putative inactivating species suggest that Lys-73, Lys-234, and Ser-130 are three likely residues that may be modified in the course of the inactivation chemistry. A discussion is presented of the mechanism of formation of the transiently inhibited enzyme species, which comes about as a consequence of the tautomerization of the double bond of the inactivating iminium moiety. In addition, the mechanistic details presented for sulbactam are compared and contrasted with those of clavulanic acid, another clinically used inactivator for class A beta-lactamases.