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 Saccharo myces cerevisiae 140-fold to electrophoretic homogeneity by a simple p rocedure involving affinity and ion exchange chromatography. The purif ied enzyme was most active at pH 6.8 and 51 degrees C. Its molecular m ass was determined to be 45000 +/- 2000 Da by SDS-polyacrylamide gel e lectrophoresis. At physiological pH values the thermodynamic equilibri um constant was determined to be 3.5 x 10(-3) (M(-1)). Product inhibit ion as well as competitive inhibition patterns were found which clearl y indicate that the kinetic mechanism of the glycerol-3-phosphate dehy drogenase is random bi-bi with the formation of dead-end complexes. In vivo concentrations of selected metabolites and kinetic expression fo r G3P-DH were used to explain regulatory properties of this enzyme und er conditions of short-term glucose effect in Saccharomyces cerevisiae .