Functional analyses of AmpC β‐lactamase through differential stability

Abstract

Despite decades of intense study, the complementarity of β‐lactams for β‐lactamases and penicillin binding proteins is poorly understood. For most of these enzymes, β‐lactam binding involves rapid formation of a covalent intermediate. This makes measuring the equilibrium between bound and free β‐lactam difficult, effectively precluding measurement of the interaction energy between the ligand and the enzyme. Here, we explore the energetic complementarity of β‐lactams for the β‐lactamase AmpC through reversible denaturation of adducts of the enzyme with β‐lactams. AmpC from Escherichia coli was reversibly denatured by temperature in a two‐state manner with a temperature of melting (T_m) of 54.6 °C and a van't Hoff enthalpy of unfolding (ΔH_VH) of 182 kcal/mol. Solvent denaturation gave a Gibbs free energy of unfolding in the absence of denaturant (ΔG_u ) of 14.0 kcal/mol. Ligand binding perturbed the stability of the enzyme. The penicillin cloxacillin stabilized AmpC by 3.2 kcal/mol (ΔT_m = + 5.8 °C); the monobactam aztreonam stabilized the enzyme by 2.7 kcal/mol (ΔT_m = +4.9 °C). Both acylating inhibitors complement the active site. Surprisingly, the oxacephem moxalactam and the carbapenem imipenem both destabilized AmpC, by 1.8 kcal/mol (ΔT_m = –3.2 °C) and 0.7 kcal/mol (ΔT_m = –1.2 °C), respectively. These β‐lactams, which share nonhydrogen substituents in the 6(7)α position of the β‐lactam ring, make unfavorable noncovalent interactions with the enzyme. Complexes of AmpC with transition state analog inhibitors were also reversibly denatured; both benzo(b)thiophene‐2‐boronic acid (BZBTH2B) and p‐nitrophenyl phenylphosphonate (PNPP) stabilized AmpC. Finally, a catalytically inactive mutant of AmpC, Y150F, was reversibly denatured. It was 0.7 kcal/mol (ΔT_m = –1.3 °C) less stable than wild‐type (WT) by thermal denaturation. Both the cloxacillin and the moxalactam adducts with Y150F were significantly destabilized relative to their WT counterparts, suggesting that this residue plays a role in recognizing the acylated intermediate of the β‐lactamase reaction. Reversible denaturation allows for energetic analyses of the complementarity of AmpC for β‐lactams, through ligand binding, and for itself, through residue substitution. Reversible denaturation may be a useful way to study ligand complementarity to other β‐lactam binding proteins as well.