FURTHER KINETIC CHARACTERIZATION OF WILD TYPE AND FEEDBACK-RESISTANT FORMS OF THE BIOSYNTHETIC sn-GLYCEROL-3-PHOSPHATE DEHYDROGENASE*

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 ubstrates about equally well by both enzymes. Both enzymes were inhibited 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 was similar to the K , for glycolaldehyde-P for both enzymes, a result unlike any other substrate-product pair tested where K , values were 20to 40-fold and 1.5to 3.7-fold higher than Ki values for the wild type and feedback-resistant enzymes, respectively. An interpretation of these data is that glycerol-P and its phosphonate analog inhibit at a glycerol-P regulatory site while ethylene glycol-P inhibits by interaction at the active site. The kinetic properties of the feedback-resistant and wild type glycerol-P dehydrogenases are remarkably similar with respect to catalytic constants but dramatically different with respect to inhibition by glycerol-P and its phosphonate analog. These kinetic data suggest a regulatory site for glycerol-P inhibition.