A single amino acid change in Escherichia coli glycerol kinase abolishes glucose control of glycerol utilization in vivo

Abstract

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...

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…A conformational change of glycerol kinase is part of the catalytic cycle, and so it was proposed that the binding of EIIA Glc could lock the protein in a closed state (355). The lower inhibition by EIIA Glc observed for some mutant GlpKs affected in the domain associated with "opening" and "closing" GlpK is consistent with this concept (655,658).…”

Transplanting allosteric control of enzyme activity by protein–protein interactions: Coupling a regulatory site to the conserved catalytic core

“…In the proposed domain-shear motion, the ␣ 1 and ␣ 2 elements move with the ␤-sheet in each domain (9). The importance of interactions between the layers is indicated by the effect of a single amino acid substitution in EcGK on IIA Glc inhibition (20). The substitution G304S alters interactions between the conserved ␤-sheet and element ␣ 1 in domain II and switches the effect of IIA Glc to activation.…”

“…Allosteric inhibition of EcGK by FBP and IIA Glc operate independently, and IIA Glc inhibition does not require tetramer formation (19)(20)(21). Thus, HiGK is naive with respect to allosteric control by E. coli IIA Glc .…”

Reverse Genetics of Escherichia coli Glycerol Kinase Allosteric Regulation and Glucose Control of Glycerol Utilization In Vivo

“…Construction and characterization of allosteric regulatory variant glycerol kinase T-477-N. Two of the allosteric variant glycerol kinases used in these studies, A-65-T and G-304-S, were described previously, and their catalytic and regulatory properties have been determined by in vitro studies with the purified enzymes (16,21). The T-477-N glycerol kinase was constructed, purified, and characterized as described in Materials and Methods.…”

“…Alleles that encode the A-65-T (glpK203) and G-304-S (glpK22) glycerol kinases were derivatives of plasmid pHG165 (35) and have been described previously (16,21). The allele for the T-477-N glycerol kinase (glpK204) was constructed by using the Kunkel method of site-directed mutagenesis, as described previously for the construction of other site-directed variants of glycerol kinase (23).…”