ESCHERICHIA-COLI GLYCEROL KINASE - ROLE OF A TETRAMER INTERFACE IN REGULATION BY FRUCTOSE-1,6-BISPHOSPHATE AND PHOSPHOTRANSFERASE SYSTEM REGULATORY PROTEIN III(GLC)

Escherichia coil glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphot ransferase) is a key element in a signal transduction pathway that cou ples expression of genes required for glycerol metabolism to the relat ive availability of glycerol and glucose. Its catalytic activity is in hibited by protein-protein interactions with IIIglc, a phosphotransfer ase system protein, and by fructose 1,6-bisphosphate (FBP); each of th ese allosteric effecters constitutes a positive signal that glucose is available. Loss of glucose inhibition of glycerol metabolism was used to screen for regulatory mutants of glycerol kinase after hydroxylami ne mutagenesis of the cloned glpK gene. Two mutant enzymes were identi fied and shown by DNA sequencing to contain the mutations alanine 65 t o threonine (A65T) and aspartate 72 to asparagine (D72N). Initial velo city studies show the mutations do not significantly affect the cataly tic properties, hence active-site structures, of the enzymes. Both mut ations decrease inhibition by FBP; A65T eliminates the inhibition whil e D72N appears to decrease the affinity for FBP and the extent of the inhibition. However, neither mutation significantly affects inhibition by IIIglc. Gel-permeation chromatography studies show that both of th e mutations after the dimer-tetramer assembly reaction of the enzyme a nd the effect of FBP in increasing the molecular weight. The effects o f the mutations on the assembly reaction are consistent with the locat ions of these two amino acid residues in the X-ray structure, which sh ows them to be associated with an alpha-helix that constitutes one of the two subunit-subunit interfaces within the tetramer. Thus, mutation s that affect the dimer-tetramer assembly of glycerol kinase decrease the regulation by FBP but do not alter regulation by IIIglc. These res ults suggest that the allosteric mechanism used to regulate the activi ty of glycerol kinase in vivo may be dependent on its concentration.