Ribose-5-phosphate isomerase from Saccharomyces cerevisiae: Purification and molecular analysis of the enzyme

Purification and molecular analysis of ribose-5-phosphate isomerase (EC5.3.1.6) from Saccharomyces cerevisiae is described first time. The enzymewas enriched from a haploid deletion mutant containing the wild-type gene ona multicopy plasmid elaborating the following steps: ammonium sulphateprecipitation, interfacial salting out on Sepharose 6B, high performanceliquid chromatography on Fractogel EMD DEAE and on Resource Phenyl. Theenzyme activity was found to be rather unstable possibly caused by removalof stabilizing cofactors or proteins during the purification procedure.The purified enzyme showed a hyperbolic dependence on the substrateribose-5-phosphate with a K_m-value of 1.6±0.3 mmol/l.For the native enzyme a molecular mass of 115±10 kDa was determinedas found by saccharose density gradient centrifugation, sedimentationequilibrium analysis, size exclusion chromatography and polyacrylamide gelelectrophoresis. Sodium dodecyl sulphate polyacrylamide gel electrophoresisand Western blotting revealed one band with a molecular mass of 31±2kDa. Thus, the native enzyme is composed of four subunits of identicalsize.The molecular mass of the subunit and the identified N-terminal sequenceof 33 amino acids fits well the 258 amino acid protein encoded by the S.cerevisiae RKI open reading frame, which was characterized previously onlyby increasing specific activities of ribose-5-phosphate isomerase in cellsafter cloning the gene. On the basis of the conserved amino acids analignment of the amino acid sequence of ribose-5-phosphate isomerase fromyeast with those of the enzyme from mouse, spinach and Escherichia coli ispresented.