Does the bifunctional uridylate synthase channel orotidine 5'-phosphate? Kinetics of orotate phosphoribosyltransferase and orotidylate decarboxylase activities fit a noninteracting sites model

Abstract

Uridylate synthase is a bifunctional protein that first forms orotidine 5'-phosphate (OMP) from orotate via its orotate phosphoribosyltransferase activity (EC 2.4.2.10) and then converts OMP to uridine 5'-phosphate (UMP) via the OMP decarboxylase activity (EC 4.1.1.23). A computer modeling analysis of the experiments that led to the proposal [Traut, T.W., & Jones, M.E. (1977) J. Biol. Chem. 252, 8374-8381] that uridylate synthase channels intermediate OMP suggests that the experimental results do not demonstrate preferential use of OMP generated in the bifunctional complex as against exogenous OMP. This analysis shows that the experimentally observed amounts of [6-14C]UMP from [6-14C]orotate in the presence of various amounts of exogenous [7-14C]OMP agree well with the amounts predicted by the computer simulations. Thus we conclude that uridylate synthase does not channel OMP. Additionally, the subsequent suggestion that channeling of OMP occurs to protect the intermediate from degradation by a nucleotidase [Traut, T.W. (1980) Arch. Biochem. Biophys. 200, 590-594] seems unlikely. The appropriate computer simulation demonstrates that low transient levels of OMP and protection of the intermediate are provided for strictly by the kinetic parameters of orotate phosphoribosyltransferase, OMP decarboxylase, and the nucleotidase. Additionally, calculations show that, in both sets of published experiments, the concentration of transient OMP greatly exceeded the concentration of OMP decarboxylase active sites. Thus, channeling of OMP by the bifunctional complex cannot be invoked to explain the evolution of uridylate synthase, and that event must be the result of some other selective pressure.

Investigation of the Mechanism of Phosphoribosylamine Transfer from Glutamine Phosphoribosylpyrophosphate Amidotransferase to Glycinamide Ribonucleotide Synthetase

“…For an excellent introduction to modeling in spreadsheet programs, see Atkinson (1987). For analogous numerical integrations of Michaelis-Menten equations, see Duggleby et al (1978), McClard and Shokat (1987), and Storer and Comish-Bowden (1974). The exact differential <51 was replaced with the interval At, which was varied in the numerical integration such that the step size was sufficiently small to accurately represent a continuous process (from 0.005 to 0.02 min).…”

Pyrimidine biosynthesis in Saccharomyces cerevisiae: the ura2 cluster gene, its multifunctional enzyme product, and other structural or regulatory genes involved in de novo UMP synthesis

“…multifunctional protein UMP synthase proposed to channel OMP (Traut and Jones 1977;McClard and Shokat 1987). However, the question still remains as to whether channeling is at work in the cell.…”