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 "P-i mop" to remove P-i from solutions and as a spectroscopic assay for P-i 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 P-i 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 P-i catalyzed by the phosphorylase limits the efficiency of the P-i mop. To overcome this, phosphodeoxyribomutase was used to catalyze the conversion of ribose-1-phosphate to ribose-5-phosphate, enabling the P-i mop to remove large amounts of P-i quantitatively, Acyclovir diphosphate provides a simple method to switch off the P-i mop as it is a tight inhibitor (K-d 12 nM) of purine nucleoside phosphorylase. (C) 1998 Academic Press.