Uridine phosphorylase from Acholeplasma laidlawii: Purification and kinetic properties

Uridine phosphorylase was purified 1,370-fold from sonicated extracts of Acholeplasma laidlawii by ammonium sulfate precipitation, DEAE-Sephadex column chromatography, hydroxylapatite chromatography, and Sephadex G-200 fractionation. The molecular weight of the enzyme as determined by gel filtration was approximately 65,000. [U-¹⁴C]ribose-1-phosphate (Rib-1-P), prepared enzymatically from [U-¹⁴C]inosine, was utilized in initial velocity studies of uridine synthesis, which indicated a sequential reaction with a K(mUra) of 110 μM and a K(mRib-1-P) of 17 μM. The kinetics of uridine cleavage was assessed at a saturating cosubstrate concentration, resulting in a K(mUrd) of 170 μM and a K(mPi) of 120 μM. These results indicate that an intracellular flux from uracil to uridine is kinetically feasible. However, such flux would be metabolically unproductive, since the low affinity of uridine kinase (K(mUrd) = 3.2 mM) precludes the operation of uridine phosphorylase and uridine kinase in tandem to convert uracil to UMP. We conclude that uridine phosphorylase performs only a catabolic function in A. laidlawii.