Studies of the binding of the magnesium-phosphoribosyl 1-pyrophosphate complex to orotate phosphoribosyltransferase from yeast

Both C14- and 13C-phosphoribosyl 1-pyrophosphate molecules, labeled at the C'-1 position, were synthesized biochemically with bacterial ribokinase and PRibPP synthetase activities. A flow dialysis experiment characterized the dissociation constant (33 μM) for the OPRTase-C14PRibPP-Mg(II) complexation and defined 2 PRibPP binding sites per enzyme dimer. Moreover, labeled-PRibPP was observed to elute with a 50 μg sample of OPRTase through Sephadex Superfine G-25, suggesting that one form of the label is bound more tightly to the enzyme then the value of Kd would suggest. Samples of 13C-PRibPP, were characterized with 13C-NMR. A doublet appeared in the proton-decoupled PRibPP spectrum. The addition of a saturating level of Mg(II) to PRibPP resulted in a 0.1 ppm upfield shift. The addition of 0.6 mM OPRTase to a sample containing 0.9 mM 13C-PRibPP and 4 mM Mg (II) resulted in a disappearance of the doublet. Upon further addition of 1 mM 13C-PRibPP, four resonances were observed and tentatively assigned as follows: beta-13C ribose 5' phosphate OPRTase bound-13C-PRibPP, alpha-13C-ribose 5' phosphate and a 13C-phosphoribosyl-OPRTase adduct. These assignments are consistent with the OPRTase ping pong kinetic mechanism, during which the pyrophosphate portion of PRibPP dissociates from the active site prior to orotate binding, and during which PRibPP undergoes hydrolysis within the time frame of the NMR experiment