title: Uridine phosphorylase from Acholeplasma laidlawii: purification and kinetic properties.
abstract: 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 KmUra of 110 microM and a KmRib-1-P of 17 microM. The kinetics of uridine cleavage were assessed at a saturating cosubstrate concentration, resulting in a KmUrd of 170 microM and a KmPi of 120 microM. 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 (KmUrd = 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.
mesh_terms: D000128:Acholeplasma laidlawii / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D010430:Pentosyltransferases / Q000378:metabolism*; D014498:Uracil / Q000378:metabolism; D014529:Uridine / Q000378:metabolism; D014541:Uridine Kinase / Q000378:metabolism; D014542:Uridine Monophosphate / Q000378:metabolism; D014543:Uridine Phosphorylase / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D014498:Uracil; D014542:Uridine Monophosphate; D010430:Pentosyltransferases; D014543:Uridine Phosphorylase; D014541:Uridine Kinase; D014529:Uridine