Effects on substrate profile by mutational substitutions at positions 164 and 179 of the class A TEM(pUC19) β-lactamase from Escherichia coli

Abstract

We investigated the effects of mutations at positions 164 and 179 of the TEM(pUC19) β-lactamase on turnover of substrates. The direct consequence of some mutations at these sites is that clinically important expanded-spectrum β-lactams, such as third-generation cephalosporins, which are normally exceedingly poor substrates for class A β-lactamases, bind the active site of these mutant enzymes more favorably. We employed site-saturation mutagenesis at both positions 164 and 179 to identify mutant variants of the parental enzyme that conferred resistance to expanded-spectrum β-lactams by their enhanced ability to turn over these antibiotic substrates. Four of these mutant variants, Arg164 → Asn, Arg164 → Set, Asp179 → Asn, and Asp179 → Gly, were purified and the details of their catalytic properties were examined in a series of biochemical and kinetic experiments. The effects on the kinetic parameters were such that either activity with the expanded-spectrum β-lactams remained unchanged or, in some cases, the activity was enhanced. The affinity of the enzyme for these poorer substrates (as defined by the dissociation constant, K(s)) invariably increased. Computation of the microscopic rate constants (k2 and k3) for turnover of these poorer substrates indicated either that the rate-limiting step in turnover was the deacylation step (governed by k3) or that neither the acylation nor deacylation became the sole rate-limiting step. In a few instances, the rate constants for both the acylation (k2) and deacylation (k3) of the extended-spectrum β-lactamase were enhanced. These results were investigated further by molecular modeling experiments, using the crystal structure of the TEM(pUC19) β-lactamase. Our results indicated that severe steric interactions between the large 7β functionalities of the expanded- spectrum β-lactams and the Ω-loop secondary structural element near the active site were at the root of the low affinity by the enzyme for these substrates. These conclusions were consistent with the proposal that the aforementioned mutations would enlarge the active site, and hence improve affinity.