Effect of divalent metal cations on the dimerization of OXA-10 and-14 class D β-lactamases from Pseudomonas aeruginosa

The factors influencing the oligomerization state of OXA-10 and OXA-14 class D beta -lactamases in solution have been investigated. Both enzymes were found to exist as an equilibrium mixture of a monomer and dieter, with a K-d close to 40 muM. The dimeric form was stabilized by divalent metal cations. The ability of different metal ions to stabilize the dimer was in the following order: Cd2+ > Cu2+ > Zn2+ > Co2+ > Ni2+ > Mn2+ > Ca2+ > Mg2+. The apparent KdS describing the binding of Zn2+ and Cd2+ cations to the OXA-10 dieter were 7.8 and 5.7 muM, respectively. The metal ions had a profound effect on the thermal stability of the protein complex observed by differential scanning calorimetry. The enzyme showed a sharp transition with a T-m of 58.7 degreesC in the absence of divalent cations, and an equally sharp transition with a T-m of 78.4 degreesC in the presence of a saturating concentration of the divalent cation. The thermal transition observed at intermediate concentrations of divalent metal ions was rather broad and lies between these two extremes of temperature. The equilibrium between the monomer and dimer is dependent on pH, and the optimum for the formation of the dimer shifted from pH 6.0 in the absence of divalent cations to pH 7.5 at saturating concentrations. The beta -lactamase activity increased approximately 2-fold in the presence of saturating concentrations of zinc and cadmium ions. Reaction with beta -lactams caused a shift in the equilibrium toward monomer formation, and thus an apparent inactivation, but the divalent cations protected against this effect.