Purification and Properties of the Flavine-Stimulated Anaerobicl-α-Glycerophosphate Dehydrogenase ofEscherichia coli

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Abstract
The anaerobicl-α-glycerophosphate (l-α-GP) dehydrogenase ofEscherichia coliwas purified approximately 40-fold. The activity of the dehydrogenase, although not affected by the addition of pyridine nucleotides, was stimulated three- to fourfold by flavine adenine dinucleotide (Kmabout 10−7m) and up to 10-fold by flavine mononucleotide (Kmabout 10−4m). Maximal activity of the enzyme was found only in the combined presence of saturating concentrations of both flavines (stimulation by a factor of 10 to 15). The dependence of the rate of the reaction on the concentration ofl-α-GP was complex in the presence of both flavines, but in the presence of flavine adenine dinucleotide alone the kinetics were of the Michaelis-Menten type with theKmforl-α-GP being about 10−4m. The product of the reaction was identified as dihydroxyacetone phosphate, and the molecular weight of the dehydrogenase was estimated to be 80,000 ± 10,000. Phenazine methosulfate, menadione and ferricyanide served as artificial acceptors for the dehydrogenase. The enzyme was sensitive to iodoacetate,p-chloromercuribenzoate, andN-ethymaleimide.