OVEREXPRESSION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT HUMAN 5-PHOSPHORIBOSYL-1-PYROPHOSPHATE SYNTHETASE ISOZYME-I AND ISOZYME-II

Human 5-phosphoribosyl-1-pyrophosphate synthetase isozymes I and II (P RSI and PRSII) have been isolated independently and characterized in p ure form. cDNAs for PRSI and PRSII were overexpressed in an Escherichi a coli strain which lacks the bacterial 5-phosphoribosyl-1-pyrophospha te synthetase. The recombinant isoforms were purified to virtual homog eneity with specific activities of 25.0 and 35.7 units/mg, respectivel y, values which are 5-10-fold higher than any previously reported for this enzyme from human sources. Despite 95% amino acid sequence identi ty, the isoforms differed significantly in several physical and kineti c properties. PRSII was more sensitive to heat inactivation at 55-degr ees-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 signific antly in K(m) values for MgATP and ribose 5-phosphate, pH optima, and Mg2+ and P(i) 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 bet ween PRSI and PRSII are consistent with the suggestion that PRSII pred ominates in rapidly proliferating cells.