Kinetic Analysis, Isotope Exchange Studies and Metal Activation Experiments carried out in this laboratory suggest that orotate phosphoribosyltransferase (O-PRTase) from Yeast may catalyze the formation of orotidine 5'monophosphate (OMP) through the use of a ping-pong kinetic mechanism but only in the presence of excess Mg2 ion. We propose to characterize this mechanism further by first detecting and then isolating the OPRTase-Phosphoribosyl intermediate that must form during OMP formation using kinetic and NMR techniques. Moreover having isolated both hypoxanthine/guanine phosphoribosyltransferase (HG-PRTase) and nicotinate phosphoribosyltransferase (N-PRTase) from this same source, we now propose to characterize the kinetic mechanisms by which guanine-, inosine-monophosphates (GMP and IMP) and nicotinate mononucleotide (NMN) are synthesized for comparison with the results obtained for the OMP synthesis. These investigations will make use of high pressure liquid chromatography (HPLC) assay procedures we have designed recently. All of these studies may reveal an intricate control mechanism, at the enzymic level, for the allocation of the phosphoribosylpyrophosphate precursor for all PRTase reactions in eukaryotic tissues.
| Ashton, R W; Strauss, R S; Chung, S H et al. (1989) Orotate phosphoribosyltransferase from yeast: studies of the structure of the pyrimidine substrate binding site. Arch Biochem Biophys 272:421-32 |
