Overexpression, purification, and characterization of recombinant human 5-phosphoribosyl-1-pyrophosphate synthetase isozymes I and II

Human 5-phosphoribosyl-1-pyrophosphate synthetase isozymes I and II (PRSI and PRSII) have been isolated independently and characterized in pure form. cDNAs for PRSI and PRSII were overexpressed in an Escherichia coli strain which lacks the bacterial 5-phosphoribosyl-1-pyrophosphate synthetase. The recombinant isoforms were purified to virtual homogeneity with specific activities of 25.0 and 35.7 units/mg, respectively, values which are 5-10-fold higher than any previously reported for this enzyme from human sources. Despite 95% amino acid sequence identity, the isoforms differed significantly in several physical and kinetic properties. PRSII was more sensitive to heat inactivation at 55 degrees C and more susceptible to disaggregation to inactive forms in the absence of Mg2+ and ATP than was PRSI. The isoforms were separable on the basis of isoelectric point. PRSI and PRSII also differed significantly in Km values for MgATP and ribose 5-phosphate, pH optima, and Mg2+ and Pi activation curves. PRSII was less sensitive to feedback inhibition by purine nucleotides and more sensitive to inhibition by 2,3-diphosphoglycerate than PRSI. Differences in kinetic properties between PRSI and PRSII are consistent with the suggestion that PRSII predominates in rapidly proliferating cells.