Leaving group activation and pyrophosphate ionic state at the catalytic site of plasmodium falciparum orotate phosphoribosyltransferase

Plasmodium falciparum orotate phosphoribosyltransferase (PfOPRT) catalyzes the reversible pyrophosphorolysis of orotidine 5′-monophosphate (OMP). Transition-state analysis from kinetic isotope effects supports a dianionic orotic acid (OA) leaving group. Isotope-edited Fourier transform infrared (FTIR) spectrometry complemented by homology modeling and quantum chemical calculations were used to characterize the orotate hydrogen-bond network for PfOPRT. Bond stretch frequencies for C₂=O and C₄=O of OMP were established from ¹³C-edited FTIR difference spectra. Both frequencies were shifted downward by 20 cm^-1 upon formation of the Michaelis complex. Hydrogen-bond interactions to the orotate moiety induce strong leaving group polarization by ground-state destabilization. The C ₂=O bond is 2.7 Å from two conserved water molecules, and the C₄=O bond is within 2.4 Å of the NH₂(ω) of Arg241 and the peptide NH of Phe97. Relative to free OMP, the N1 atom of PfOPRT-bound OMP indicates a ΔpK_a of -4.6. The decreased basicity of N1 supports leaving group activation through a hydrogen-bond network at the PfOPRT active site. PfOPRT in complex with ¹⁸O-PPi and a proposed transition-state analogue revealed a trianionic PPi nucleophile with no significant P••O bond polarization, supporting a mechanism proceeding through the migration of the ribocation toward the PPi. These results along with previous PfOPRT transition-state analyses provide reaction coordinate information for the PfOPRT-catalyzed OMP pyrophosphorolysis reaction.