Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase) is a key element in glucose control of glycerol metabolism. Its catalytic activity is inhibited allosterically by the glycolytic intermediate, fructose 1,6-bisphosphate, and by the phosphotransferase system phosphocarrier protein, III Glc (also known as IIA Glc ). These inhibitors provide mechanisms by which glucose blocks glycerol utilization in vivo. We report here the cloning and sequencing of the glpK22 gene isolated from E. C. C. Lin strain 43, a strain that shows the loss of glucose control of glycerol utilization. DNA sequencing shows a single missense mutation that translates to the amino acid change Gly-304 to Ser (G-304-S) in glycerol kinase. The effects of this substitution on the functional and physical properties of the purified mutant enzyme were determined. Neither of the allosteric ligands inhibits it under conditions that produce strong inhibition of the wild-type enzyme, which is sufficient to explain the phenotype of strain 43. However, III Glc activates the mutant enzyme, which could not be predicted from the phenotype. In the wild-type enzyme, G-304 is located 1.3 nm from the active site and 2. Glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase) catalyzes the rate-limiting step in glycerol utilization by Escherichia coli (25). Its catalytic activity is regulated at the protein level by inhibition by two allosteric effectors, the glucose-specific phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system, III Glc (also known as IIA Glc ) (13, 18), and the glycolytic intermediate, fructose 1,6-bisphosphate (FBP) (26). Control by III Glc is dependent on its state of phosphorylation in a mechanism termed inducer exclusion, which has been reviewed recently (12,19,20). These effectors provide the basis for glucose inhibition of glycerol utilization. In wild-type cells, this inhibition prevents synthesis of glycerol 3-phosphate, which is the inducer for the elements of the glp regulon. We are investigating the molecular basis of these allosteric control mechanisms by which glycerol kinase functions in a signal transduction pathway that modulates gene expression in response to carbon source availability. The crystal structure of the complex of glycerol kinase with the unphosphorylated form of III Glc has been determined (8), and the association of the two proteins forms a novel intermolecular binding site for Zn(II) (5). A proposed mechanism for FBP regulation postulates that FBP binds to and inhibits the tetrameric form of the enzyme (3). We have recently shown that amino acid substitutions in the tetramer interface which decrease the extent of tetramer formation also decrease FBP inhibition and that FBP and III Glc inhibition can operate independently (10).We report here the cloning and sequencing of the glpK22 gene from E. C. C. Lin strain 43. The phenotype for this mutant is the loss of glucose inhibition of glycerol utilization, resulting in simultaneous utilization of both carb...