Cloning, Overproduction, and Purification of Native and Mutant Recombinant Yeast Orotate Phosphoribosyltransferase and the Demonstration from Magnetization Inversion Transfer That a Proposed Oxocarbocation Intermediate Does Not Have a Kinetic Lifetime

The gene for orotate phosphoribosyltransferase fromSaccharomyces cerevisiaehas been subcloned into anEscherichia colioverexpression vector and the enzyme has been produced in large quantities, thus simplifying the purification to one step. We were able to repeat the published (J. Victor, L. B. Greenberg, and D. L. SloanJ. Biol. Chem.254, 2647–2655, 1979).32PPi/5-phosphorylribose 1-α-diphosphate exchange experiments and could demonstrate the exchange by magnetization inversion transfer NMR experiments as well. However, when contaminating orotidine 5′-monophosphate (OMP) was eliminated with OMP decarboxylase, any evidence of magnetization transfer vanished. Consequently, it is concluded that a ping pong mechanism is not operable and that a previously proposed oxocarbocation intermediate along the pathway to OMP does not persist long enough in the catalytic cycle of this enzyme to be recognized by NMR exchange experiments.