Kinetic and conformational studies of the orotate phosphoribosyltransferase:orotidine-5'-phosphate decarboxylase enzyme complex from mouse Ehrlich ascites cells.

Complex U is composed of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine-5’-phosphate decarboxylase (EC 4.1.1.23), the last two enzymes of the de nouo pathway for pyrimidine biosynthesis. Since the two enzymes have proved inseparable so far, the equilibrium constant for the phosphoribosyltransferase activity has been determined under conditions where the decarboxylase activity was totally inhibited by B-azaUMP. The K,, for orotate phosphoribosyltransferase in the direction of orotidine-5’phosphate (OMP) synthesis is 0.07. Therefore, the coupling of this reversible reaction with the irreversible decarboxylase reaction would seem to be important in promoting the synthesis of UMP. The possibility that the enzyme complex might “channel” the product of the phosphoribosyltransferase, OMP (without liberating it into solution), to UMP was tested with an assay procedure that can recover and measure the substrate, orotate, as well as OMP and UMP by thin layer chromatography. These experiments were done by adding various concentrations of OMP to reaction mixtures which contained orotate and phosphoribosyl pyrophosphate as substrates. The addition of OMP, the product of the phosphoribosyltransferase, increases the activity of the phosphoribosyltransferase. This activation is maximum when the added OMP pool is 10 PM. Our results indicate that strict channeling of complex-formed OMP to UMP does not occur in the presence of a large exogenous OMP pool; however, some channeling occurs at all concentrations of OMP, and considerable channeling occurs at low exogenous OMP concentrations. Therefore, complex U can convert orotate to UMP so smoothly that OMP synthesized by the enzyme complex only partially equilibrates with a pool of exogenous OMP and is preferentially decarboxylated by the decarboxylase of complex U. We interpret these results as evidence of partial channeling by the enzyme complex. The activation of the phosphoribosyltransferase activity