Characterization of a PSE-4 mutant with different properties in relation to penicillanic acid sulfones: Importance of residues 216 to 218 in class A β-lactamases

Abstract

Class A β-lactamases are inactivated by the suicide inactivators sulbactam, clavulanic acid, and tazobactam. An examination of multiple alignments indicated that amino acids 216 to 218 differed among class A enzymes. By random replacement mutagenesis of codons 216 to 218 in PSE-4, a complete library consisting of 40,864 mutants was created. The library of mutants with mutations at positions 216 to 218 in PSE-4 was screened on carbenicillin and ampicillin with the inactivator sulbactam; a collection of 14 mutants was selected, and their bla genes were completely sequenced. Purified wild-type and mutant PSE-4 β-lactamases were used to measure kinetic parameters. One enzyme, V216S:T217A:G218R, was examined for its peculiar pattern of inhibition. There was an increase in the K(m) from 68 μM for the wild type to 271 μM for the mutant for carbenicillin and 33 to 216 μM for ampicillin. Relative to the wild-type PSE-4 enzyme, 37- and 30-fold increases in K(i) values were observed for the mutant enzyme for sulbactam and tazobactam, respectively. The results that were obtained suggested that positions 216 to 218 are important for interactions with penicillanic acid sulfone inhibitors. In contrast, V216 and A217 in the TEM-1 class A β- lactamase do not tolerate amino acid residue substitutions. However, for the PSE-4 β-lactamase, 11 of 14 mutants from the library of mutants with mutations at positions 216 to 218 whose sequences were determined had substitutions at position 216 (G, R, A, S) and position 217 (A, S). The data showed the importance of residues 216 to 218 in their atomic interactions with inactivators in the PSE-4 β-lactamase structure.