Enzymatic kinetic analyses that employ high-performance liquid chromatography. Competition between orotate- and...

Enzymatic assay procedures that employ high-performance liquid chromatography (HPLC) have been proven to be sensitive and versatile methods for accomplishing kinetic analyses of enzyme-catalyzed reactions, with nucleotides as substrates or products. Both orotate phosphoribosyltransferase (OPRTase) and hypoxanthine/guanine phosphoribosyltransferase (HGPRTase) have been purified from Baker's yeast and analyzed kinetically using a modification of published HPLC procedures. Because these two enzymes exist in the cytosol of yeast and might compete for the limiting (approximately equal to 15 microM) concentration of phosphoribosyl alpha-1-pyrophosphate (PRibPP), we elected to examine both equilibrium and steady-state effects of one enzymatic reaction on the other with HPLC. First, under the condition of equivalent mass concentrations of OPRTase and HGPRTase, the initial rate of orotidine monophosphate synthesis and the equilibrium state were greatly affected by the presence of HGPRTase activity. In contrast, the presence of the OPRTase activity had no effect on the HGPRTase-catalyzed reaction under these conditions. Second, to examine a competition by these enzymes for PRibPP in vivo, we have established that the total activities (units/ml) of OPRTase and HGPRTase in yeast cell extracts were 740 units/ml and 450 units/ml, respectively (a 1.7:1 ratio). These relative activities were then employed in an in vitro reaction competition analysis. The results were similar to the those obtained from experiments where equivalent OPRTase and HGPRTase activities were employed and reveal profound initial velocity and equilibrium effects of one reaction on the other. Thus a real competition between these enzymes for PRibPP may occur in the yeast cell cytosol, as determined by this unique HPLC competition assay procedure.