Theoretical determination of two structural forms of the active site in cadmium-containing phosphotriesterases

Two structural forms of the active site in Cd²⁺-containing phosphotriesterases (PTE), i.e., Cd²⁺/Cd²⁺-PTE and Zn²⁺/Cd²⁺-PTE, were determined by performing ab initio and density functional theory calculations on active site models. The calculated results indicate that the second bridging ligand in the active site of the recently reported high-resolution X-ray crystal structures of Cd²⁺/Cd²⁺-PTE and Zn²⁺/Cd²⁺-PTE should be a hydroxide ion, rather than a water molecule. The protonated active site structures of the Cd²⁺-containing enzymes are all significantly different from that of Zn²⁺/Zn²⁺-PTE. In Cd²⁺/Cd²⁺-PTE and Zn²⁺/Cd²⁺PTE, the second bridging ligand always simultaneously coordinates to the two metal ions, whether it is a hydroxide ion or a water molecule. The pK_a values calculated for the second bridging ligand are in good agreement with the observed kinetic pK_a values for both the Cd²⁺- and Zn²⁺-substituted enzymes. The calculated results strongly support the conclusion that the observed kinetic pK_a is actually the pK_a of the second bridging ligand, and strongly support the previously postulated catalytic mechanism of PTE involving a bridging hydroxide that attacks the phosphorus center of the substrate.