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 (Tm) of 54.6 °C and
a van't Hoff enthalpy of unfolding (ΔHVH)
of 182 kcal/mol. Solvent denaturation gave a Gibbs free
energy of unfolding in the absence of denaturant
(ΔGuH2O)
of 14.0 kcal/mol. Ligand binding perturbed
the stability of the enzyme. The penicillin cloxacillin
stabilized AmpC by 3.2 kcal/mol (ΔTm
= +5.8 °C); the monobactam aztreonam stabilized the
enzyme by 2.7 kcal/mol (ΔTm = +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 (ΔTm
= −3.2 °C) and 0.7 kcal/mol (ΔTm
= −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 (ΔTm = −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.