Effects on substrate profile by mutational substitutions at positions 164 and 179 of the class A TEMpUC19 beta-lactamase from Escherichia coli

We investigated the effects of mutations at positions 164 and 179 of the TE RpUC19 beta-lactamase on turnover of substrates, The direct consequence of some mutations at these sites is that clinically important expanded-spectru m p-lactams, such as third-generation cephalosporins, which are normally ex ceedingly poor substrates for class A p-lactamases, bind the active site of these mutant enzymes more favorably. We employed site-saturation mutagenes is at both positions 164 and 179 to identify mutant variants of the parenta l enzyme that conferred resistance to expanded-spectrum p-lactams by their enhanced ability to turn over these antibiotic substrates, Four of these mu tant variants, Arg(164) --> Asn, Arg(164) --> Ser, Asp(179) --> Asn, and As p(179), Gly, were purified and the details of their catalytic properties we re examined in a series of biochemical and kinetic experiments. The effects on the kinetic parameters were such that either activity with the expanded -spectrum p-lactams remained unchanged or, in some cases, the activity was enhanced. The affinity of the enzyme for these poorer substrates (as define d by the dissociation constant, K-s) invariably increased, Computation of t he microscopic rate constants (k(2) and k(3)) for turnover of these poorer substrates indicated either that the rate-limiting step in turnover was the deacylation step (governed by k(3)) or that neither the acylation nor deac ylation became the sole rate-limiting step. In a few instances, the rate co nstants for both the acylation (k(2)) and deacylation (k(3)) of the extende d-spectrum p-lactamase were enhanced. These results were investigated furth er by molecular modeling experiments, using the crystal structure of the TE MpUC19 beta-lactamase, Our results indicated that severe steric interaction s between the large 7 beta functionalities of the expanded-spectrum beta-la ctams and the Omega-loop secondary structural element near the active site were at the root of the low affinity by the enzyme for these substrates. Th ese conclusions were consistent with the proposal that the aforementioned m utations would enlarge the active site, and hence improve affinity.