C-13 NMR-STUDIES AND PURIFICATION OF GLUCONATE PATHWAY ENZYMES FROM SCHIZOSACCHAROMYCES-POMBE

Evidence is presented to show that D-glucose in Schizosaccharomyces po mbe can be metabolized via a new alternative route (gluconate pathway) in addition to the regular D-glucose B-phosphate route, This gluconat e pathway consists of two steps: oxidation of D-glucose to D-gluconate by NADP(+)-dependent glucose dehydrogenase and phosphorylation of D-g luconate to D-phosphogluconate by gluconate kinase, The formation of D -gluconate and 6-phosphogluconate from D-glucose was monitored by C-13 nuclear magnetic resonance spectroscopy using DE [1-C-13]glucose and D-[U-C-13]glucose. The operation of the gluconate pathway was further substantiated by the purification of its two member enzymes, glucose d ehydrogenase and gluconate kinase, from the cell-free extract of the f ission yeast. Glucose dehydrogenase has been purified (580-fold) to ho mogeneity by the combined procedures of ammonium sulfate fractionation , Sephadex gel filtration, cation-exchange chromatography, matrex gel chromatography, and agarose-NADP(+) affinity chromatography, The purif ied enzyme is monomeric with a relative molecular weight of 6.65 X 10( 4) Da. Gluconate kinase has been purified (410-fold) to near homogenei ty by a combination of chromatographic procedures using Biogels, matre x gel, and agarose gels, The purified enzyme is monomeric with a relat ive molecular weight of 2.4 X 10(4) Da. The gluconate pathway presente d here provides an alternative route for the D-glucose metabolism in S ch. pombe, Meanwhile, this paper documents another metabolic differenc e between the fission and budding yeasts. (C) 1995 Academic Press, Inc .