Improved purification of ribulose 5-phosphate 3-epimerase from Saccharomyces cerevisiae and characterization of the enzyme.

D-Ribulose 5-phosphate 3-epimerase from Saccharomyces cerevisiae was purified to homogeneity by 1970-fold enrichment elaborating the following steps: disruption of fresh cells, polyethylene glycol precipitation, ion exchange chromatography, heat-treatment, size exclusion chromatography on Sephadex G-75 and Bio-Sil SEC 125 and hydrophobic interaction chromatography. A molecular mass of 50 +/- 4 kDa was determined for the native enzyme by sedimentation equilibrium experiments. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a single band with 26 kDa which has been characterized as an individual polypeptide chain. Thus, the enzyme is a dimer composed of two identical subunits. The specific activity of the purified enzyme with 7700 units/mg protein was found to be 30-fold higher than described in previous papers. The enzyme shows a hyperbolic dependence of the catalytic activity towards ribulose 5-phosphate with a KM-value of 1.5 mmol/l. The N-terminal amino acid sequence analysis of the native enzyme and of several peptides obtained by chemical and proteolytic fragmentation provided a part of the primary structure which fits to the primary structure deduced from the DNA sequence.