Purification and regulatory properties of the biosynthetic L-glycerol 3-phosphate dehydrogenase from Escherichia coli.

The L-glycerol-3-phosphate: NAD(P) oxidoreductase (EC 1.1.1.8). referred to as glycerol phosphate dehydrogenase, was purified from extracts of Escherichia coli by a combination of chromatographic procedures. The over-all purification was approximately lOOO-fold and the yield was about 10%. The elution profile from the final DEAE-Sephadex column and zone electrophoresis indicated that the enzyme preparation was not homogenous. Kinetic studies showed that the enzyme was both stabilized and inhibited by 0.1 to 1.0 M salts, and that L-glycerol 3-phosphate specifically inhibited dihydroxyacetone phosphate reduction. The curve describing the degree of inhibition as a function of Lglycerol 3-phosphate concentration was sigmoid, with 35 PM glycerol j-phosphate producing 50% inhibition. Michaelis-Menten type kinetics were found with glycerol 3-phosphate, acting as a substrate, dihydroxyacetone phosphate, and TPNH. The calculated Km values were 210 w, 170 PM, and 10 PM, respectively. Glycerol 3-phosphate was a competitive inhibitor with respect to dihydroxyacetone phosphate and an uncompetitive inhibitor with respect to TPNH. The addition of TPNH to the enzyme solution quenched the fluorescence of the protein at 340 rnp and enhanced the TPNH fluorescence at 450 rnp. Glycerol 3-phosphate further enhanced the fluorescence of the TPNHenzyme complex in the 430 to 440 mp region. The fluorimetric titration of the TPNH-enzyme complex with glycerol d-phosphate gave a sigmoid curve with the same characteristics as the inhibition curve. The inhibition of the pyridine nucleotide-linked glycerol phosphate dehydrogenase of Escherichia coli, whose most likely function is to provide glycerol 3-phosphate for lipid syntheses, could act in the cell as an effective mechanism for the restriction of the pool size of glycerol phosphate.