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-biphosphate, and by the phosphotransferase system phosphocarrier protein, IIIGlc (also known as IIAGlc). 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, IIIGlc 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 2.5 nm from the IIIGlc binding site (M. Feese, D. W. Pettigrew, N. D. Meadow, S. Roseman, and S. J. Remington, Proc. Natl. Acad. Sci. USA 91:3544-3548, 1994). It is located in the same region as amino acid substitutions in the related protein DnaK which alter its catalytic and regulatory properties and which are postulated to interfere with a domain closure motion (A. S. Kamath-Loeb, C. Z. Lu, W.-C. Suh, M. A. Lonetto, and C. A. Gross, J. Biol. Chem. 270:30051-30059, 1995). The global effect of the G-304-S substitution on the conformation and catalytic and regulatory properties of glycerol kinase is consistent with a role for the domain closure motion in the molecular mechanism for glucose control of glycerol utilization.