The PGM3 gene encodes the major phosphoribomutase in the yeast Saccharomyces cerevisiae

► Pgm1, Pgm2, and Pgm3 accept glucose-1-P and ribose-1-P as substrates in vitro. ► Ribose-1-P is formed in vivo upon perturbation of energetic equilibrium. ► Ribose-1-P only hyper-accumulates in mutants carrying a deletion in PGM3. ► Pgm3 is the major phosphoribomutase in yeast. The phosphoglucomutases (PGM) Pgm1, Pgm2, and Pgm3 of the yeast Saccharomyces cerevisiae were tested for their ability to interconvert ribose-1-phosphate and ribose-5-phosphate. The purified proteins were studied in vitro with regard to their kinetic properties on glucose-1-phosphate and ribose-1-phosphate. All tested enzymes were active on both substrates with Pgm1 exhibiting only residual activity on ribose-1-phosphate. The Pgm2 and Pgm3 proteins had almost equal kinetic properties on ribose-1-phosphate, but Pgm2 had a 2000 times higher preference for glucose-1-phosphate when compared to Pgm3. The in vivo function of the PGMs was characterized by monitoring ribose-1-phosphate kinetics following a perturbation of the purine nucleotide balance. Only mutants with a deletion of PGM3 hyper-accumulated ribose-1-phosphate. We conclude that Pgm3 functions as the major phosphoribomutase in vivo.