Purine Nucleoside Phosphorylase: Its Use in a Spectroscopic Assay for Inorganic Phosphate and for Removing Inorganic Phosphate with the Aid of Phosphodeoxyribomutase

Abstract

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 “Pimop” to remove Pifrom solutions and as a spectroscopic assay for Piat micromolar concentrations. An expression system was developed for the phosphorylase fromEscherichia 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−1were obtained for the phosphorolysis at 30°C, so that when the phosphorylase is coupled to the phosphatase being studied, rates of Pirelease 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 Picatalyzed by the phosphorylase limits the efficiency of the Pimop. To overcome this, phosphodeoxyribomutase was used to catalyze the conversion of ribose-1-phosphate to ribose-5-phosphate, enabling the Pimop to remove large amounts of Piquantitatively. Acyclovir diphosphate provides a simple method to switch off the Pimop as it is a tight inhibitor (Kd12 nM) of purine nucleoside phosphorylase.