CHARACTERIZATION OF A PSE-4 MUTANT WITH DIFFERENT PROPERTIES IN RELATION TO PENICILLANIC ACID SULFONES - IMPORTANCE OF RESIDUE-216 TO RESIDUE-218 IN CLASS-A BETA-LACTAMASES

Class A beta-lactamases are inactivated by the suicide inactivators su lbactam, clavulanic acid, and tazobactam. An examination of multiple a lignments indicated that amino acids 216 to 218 differed among class A enzymes. By random replacement mutagenesis of codons 216 to 218 in PS E-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 sulbact am; a collection of 14 mutants was selected, and their bla genes were completely sequenced. Purified wild-type and mutant PSE-4 beta-lactama ses were used to measure kinetic parameters. One enzyme, V216S:T217A:G 218R, was examined for its peculiar pattern of inhibition. There was a n increase in the K-m from 68 mu M for the wild type to 271 mu M for t he mutant for carbenicillin and 33 to 216 mu M for ampicillin. Relativ e to the wild-type PSE-4 enzyme, 37- and 30-fold increases in K-i valu es were observed for the mutant enzyme for sulbactam and tazobactam, r espectively. The results that were obtained suggested that positions 2 16 to 218 are important for interactions with penicillanic acid sulfon e inhibitors. In contrast, V216 and A217 in the TEM-1 class A beta-lac tamase do not tolerate amino acid residue substitutions. However, for the PSE-4 beta-lactamase, If of 14 mutants from the library of mutants with mutations at positions 216 to 218 whose sequences were determine d 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 ato mic interactions with inactivators in the PSE-4 beta-lactamase structu re.