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.