Homogeneous wild type and feedback-resistant forms of the biosynthetic glycerol-3-phosphate dehydrogenase (NAD+) of Escherichia coli (EC 1.1.1.8) were employed for studies of substrate and inhibitor specificity. The phosphonate analog of dihydroxyacetone-P, 4-hydroxy-3-oxybutyl 1-phosphonate, and glycolaldehyde phosphate proved to be substrates of both enzymes. NADPH, NADH, and nicotinamide hypoxanthine dinucleotide were used as substrates about equally well by both enzymes. Both enzymes were

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... hibited to the same degree by a number of compounds which were competitive inhibitors with respect to NADPH. The enzymes were shown to have B-type stereospecificity for NADPH. All of these kinetic characterizations indicate that the active sites of the two enzymes are virtually identical. The phosphonate analog of glycerol-P, 3,4-dihydroxybutyl l-phosphonate, resembled glycerol-P in that it was a competitive inhibitor with respect to dihydroxyacetone-P and its Ki was greater than 10-fold higher for the feedback-resistant than the wild type glycerol-P dehydrogenase. The Ki values for ethylene glycol-P were similar (about 1.4 mM) for both enzymes. The Ki for the ethylene glycol-P