A NEW TEM BETA-LACTAMASE DOUBLE MUTANT WITH BROADENED SPECIFICITY REVEALS SUBSTRATE-DEPENDENT FUNCTIONAL INTERACTIONS

Using a random combinatorial mutagenesis of TEM beta-lactamase, direct ed against residues potentially involved in substrate discrimination, followed by selection on third generation cephalosporins, we obtained the double mutant E104M/G238S. Additionally, by using cloning strategi es and site-directed mutagenesis we constructed the individual single mutants and also the single modification E104K and the double mutant E 104K/G238S, which broaden the specificity of clinically isolated TEM b eta-lactamase variants. The kinetic characterization of the purified d ouble mutant E104M/G238S and its single counterparts E104M and G238S w as carried out. The single mutant E104M exhibited increased k(cat) val ues against all substrates tested. K-m values remained similar to the values shown by the wild-type enzyme. The mutation at E104M was respon sible for the increased hydrolysis rate against cefuroxime shown by th e double mutant E104M/G238S. The effect of mutation G238S varied more pronouncedly, depending on the substrate. In general, a lower K-m was observed, but also a decreased k(cat). The double mutant E104M/G238S e xhibited a higher hydrolytic rate against cefotaxime compared with the corresponding single mutations. We observed nearly a 1000-fold greate r k(cat)/K-m for the double mutant than for the wild type. This improv ement in catalysis was the consequence of increased k(cat) and decreas ed K-m values. Computed contact interactions from modeling substrate c omplexes show reliable results only for benzylpenicillin. The modeling results with this substrate confirmed the observed enzyme activities for the different single and double mutants. Analysis of the apparent coupling energies, as calculated from the kinetic parameters of the si ngle and double mutants, showed that the quantitative effect of a seco nd mutation on a single mutant was either absent, additive, partially additive, or synergistic with respect to the first mutation, depending on the substrate analyzed.