Carbon-13 NMR studies and purification of gluconate pathway enzymes from Schizosaccharomyces pombe.
Evidence is presented to show that D-glucose in Schizosaccharomyces pombe can be metabolized via a new alternative route (gluconate pathway) in addition to the regular D-glucose 6-phosphate route. This gluconate pathway consists of two steps: oxidation of D-glucose to D-gluconate by NADP(+)-dependent glucose dehydrogenase and phosphorylation of D-gluconate to 6-phosphogluconate by gluconate kinase. The formation of D-gluconate and 6-phosphogluconate from D-glucose was monitored by 13C nuclear magnetic resonance spectroscopy using D-[1-13C]glucose and D-[U-13C]glucose. The operation of the gluconate pathway was further substantiated by the purification of its two member enzymes, glucose dehydrogenase and gluconate kinase, from the cell-free extract of the fission yeast. Glucose dehydrogenase has been purified (580-fold) to homogeneity by the combined procedures of ammonium sulfate fractionation, Sephadex gel filtration, cation-exchange chromatography, matrex gel chromatography, and agarose-NADP+ affinity chromatography. The purified enzyme is monomeric with a relative molecular weight of 6.65 x 10(4) Da. Gluconate kinase has been purified (410-fold) to near homogeneity by a combination of chromatographic procedures using Bio-gels, matrex gel, and agarose gels. The purified enzyme is monomeric with a relative molecular weight of 2.4 x 10(4) Da. The gluconate pathway presented here provides an alternative route for the D-glucose metabolism in Sch. pombe. Meanwhile, this paper documents another metabolic difference between the fission and budding yeasts.
Department of Chemistry, Carleton University, Ottawa, Ontario, Canada.
C S
CS
Tsai
H G
HG
Ye
J L
JL
Shi
D002247:Carbon Isotopes; D005942:Gluconates / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D042843:Glucose 1-Dehydrogenase; D005948:Glucose Dehydrogenases / Q000302:isolation & purification; D009682:Magnetic Resonance Spectroscopy; D008954:Models, Biological; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000302:isolation & purification; D012568:Schizosaccharomyces / Q000201:enzymology / Q000378:metabolism*; D013403:Sugar Phosphates / Q000378:metabolism*
D002247:Carbon Isotopes; D005942:Gluconates; D013403:Sugar Phosphates; D005948:Glucose Dehydrogenases; D042843:Glucose 1-Dehydrogenase; D017853:Phosphotransferases (Alcohol Group Acceptor); C020578:gluconokinase; D005947:Glucose; C030691:gluconic acid; C008884:6-phosphogluconic acid