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 C(2)═O and C(4)═O of OMP were established from (13)C-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 C(4)═O bond is within 2.4 Å of the NH(2)(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 (18)O-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.
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, United States.
Yong
Y
Zhang
Hua
H
Deng
Vern L
VL
Schramm
D020134:Catalytic Domain; D011756:Diphosphates / Q000737:chemistry*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D009962:Orotate Phosphoribosyltransferase / Q000737:chemistry* / Q000378:metabolism; D009963:Orotic Acid / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry; D010963:Plasmodium falciparum / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D017550:Spectroscopy, Fourier Transform Infrared; D055672:Static Electricity
D011756:Diphosphates; D012996:Solutions; D009963:Orotic Acid; D009962:Orotate Phosphoribosyltransferase; D010100:Oxygen