Baker's yeast UMP:pyrophosphate phosphoribosyltransferase. Purification, enzymatic and kinetic properties.

Baker’s yeast UMP:pyrophosphate phosphoribosyltransferase (EC 2.4.2.9) has been extensively purified and the final preparation seemed to be pure but not homogeneous. The native enzyme showed a molecular weight of 80,000 calculated by gel filtration; disc gel electrophoresis performed in the presence of sodium dodecyl sulfate and reducing agents showed two dissimilar subunits and the sum of their respective molecular weights was in good agreement with the value obtained for the native protein. Isoelectric focusing and acrylamide gel electrophoresis demonstrated the presence of two different forms of enzyme. The enzyme is active from pH 6.5 to 10 with maximum activity at pH 7.8. UMP:pyrophosphate phosphoribosyltransferase displays linear kinetics and a family of parallel lines was generated when the reciprocal of initial velocity of UMP formation was plotted as a function of either the reciprocal of uracil concentrations or concentrations of the dimagnesium salt of 5-phosphoribosyl-1-pyrophosphate (MgzPP-ribose-P). The K, values for uracil and MgzPP-ribose-P were 21.0 PM and 26.0 PM, respectively. Among a variety of compounds tested, UMP, dUMP, dCMP, UDP, TTP, and dCTP allosterically inhibited the enzyme activity. The enzyme was highly unstable and it could be purified using dimethyl sulfoxide as a stabilizing agent. Furthermore, the activity rapidly disappeared by heating at 60°C; this inactivation could be partially prevented by the presence of some effecters.