Purine nucleoside phosphorylase: its use in a spectroscopic assay for inorganic phosphate and for removing inorganic phosphate with the aid of phosphodeoxyribomutase.
The kinetics of the phosphorolysis of 7-methylated guanosine analogues catalyzed by purine nucleoside phosphorylase has been analyzed to understand the use of this system as a "Pi mop" to remove Pi from solutions and as a spectroscopic assay for Pi at micromolar concentrations. An expression system was developed for the phosphorylase from Escherichia coli: this protein (subunit molecular mass 26 kDa) and one from a commercial source (29 kDa) were used in this study. Rates of >50 s-1 were obtained for the phosphorolysis at 30 degrees C, so that when the phosphorylase is coupled to the phosphatase being studied, rates of Pi release from the phosphatase can be measured close to this rate. The kinetic mechanism appears to obey the Michaelis-Menten model in the steady state with the bond cleavage rate limiting. Slow hydrolysis of ribose-1-phosphate to Pi catalyzed by the phosphorylase limits the efficiency of the Pi mop. To overcome this, phosphodeoxyribomutase was used to catalyze the conversion of ribose-1-phosphate to ribose-5-phosphate, enabling the Pi mop to remove large amounts of Pi quantitatively. Acyclovir diphosphate provides a simple method to switch off the Pi mop as it is a tight inhibitor (Kd 12 nM) of purine nucleoside phosphorylase.
Nixon
A E
AE
National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK.
Hunter
J L
JL
Bonifacio
G
G
Eccleston
J F
JF
Webb
M R
MR
D000212:Acyclovir / Q000494:pharmacology; D002384:Catalysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D010710:Phosphates / Q000032:analysis*; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D004791:Enzyme Inhibitors; D010710:Phosphates; D011683:Purine-Nucleoside Phosphorylase; D010770:Phosphotransferases; C022375:phosphopentomutase; D000212:Acyclovir