EFFECT OF AN AMINO-ACID INSERTION INTO THE OMEGA-LOOP REGION OF A CLASS-C BETA-LACTAMASE ON ITS SUBSTRATE-SPECIFICITY

The extended-substrate specificity of Enterobacter cloacae GC1 beta-la ctamase is entirely due to a three amino acid insertion after position 207. To clarify the reason for the extended-substrate specificity, Al a, Ala-Ala, Ala-Ala-Ala, and Ala-Ala-Ala-Ala were inserted after posit ion 207 on the basis of the class C beta-lactamase from E. cloacae P99 , respectively. The k(cat) and K-m values of all the mutant enzymes fo r cephalothin, benzylpenicillin and ampicillin were almost the same as those of the wild-type enzyme, except for those of P99-210-4A which w ere decreased 4-15-fold. On the other hand, the k(cat) and K-m values for oxyimino beta-lactams such as cefuroxime, ceftazidime, and aztreon am increased with increasing numbers of inserted alanines. The k(cat) values of the mutant enzymes for cefroxime increased 140-7400-fold com pared with that of the wild-type. The K-m, values also increased with almost the same magnitude, resulting in about the same k(cat)/K-m valu es as that of the wild-type. On progressive inhibition analysis of azt reonam of the mutant enzymes, two kinds of inactive acyl-enzyme with d istinct stabilities were observed, and the proportion of the less stab le inactive enzyme increased with increasing numbers of inserted alani nes. This suggests that the extension of the substrate specificity is due to instability of the acyl-intermediate caused by an increased dea cylation rate in the reaction process.