Fast purification and kinetic studies of the glycerol-3-phosphate dehydrogenase from the yeast Saccharomyces cerevisiae.
The glycerol-3-phosphate dehydrogenase has been purified from Saccharomyces cerevisiae 140-fold to electrophoretic homogeneity by a simple procedure involving affinity and ion exchange chromatography. The purified enzyme was most active at pH 6.8 and 51 degrees C. Its molecular mass was determined to be 45000 +/- 2000 Da by SDS-polyacrylamide gel electrophoresis. At physiological pH values the thermodynamic equilibrium constant was determined to be 3.5 x 10(-3) (M-1). Product inhibition as well as competitive inhibition patterns were found which clearly indicate that the kinetic mechanism of the glycerol-3-phosphate dehydrogenase is random bi-bi with the formation of dead-end complexes. In vivo concentrations of selected metabolites and kinetic expression for G3P-DH were used to explain regulatory properties of this enzyme under conditions of short-term glucose effect in Saccharomyces cerevisiae.
Cai
J
J
Institut für Bioverfahrenstechnik, Universität Stuttgart, Germany.
Pietzsch
M
M
Theobald
U
U
Rizzi
M
M
D001667:Binding, Competitive; D005947:Glucose / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D005993:Glycerolphosphate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D009243:NAD / Q000378:metabolism / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000201:enzymology*; D013379:Substrate Specificity
D009243:NAD; D005993:Glycerolphosphate Dehydrogenase; D005947:Glucose; D005990:Glycerol