Catalytic and Allosteric Properties of Glycerol Kinase from Escherichia coli

Abstract

Abstract Catalytic and allosteric properties of the normal and of a genetically altered glycerol kinase from Escherichia coli were examined. Normal glycerol kinase exhibited Michaelis-Menten kinetics for glycerol with an apparent Km of 10 µm. Gel filtration studies indicated that, at saturation, 3.7 moles of [14C]glycerol bound per mole of enzyme, and a dissociation constant of 10 µm was determined by ultrafiltration. The response of reaction velocity to MgATP concentration was not hyperbolic, and double reciprocal plots displayed downward curvature, indicating two apparent Km values of 80 to 100 µm and 400 to 500 µm. The unusual MgATP kinetic data were obtained by two different assay procedures, were observed throughout the course of purification, were the same for different enzyme preparations, and were unchanged by aging of the enzyme. Product inhibition studies suggested an ordered mechanism for the enzyme, with glycerol as the first substrate to bind. Fructose 1,6-bisphosphate was a noncompetitive inhibitor with respect to both substrates. The response of reaction velocity to fructose 1,6-bisphosphate concentration was slightly sigmoid, maximum inhibition was about 80%, and double reciprocal plots of fractional inhibition against inhibitor concentration indicated an apparent Ki of 0.5 mm. Gel filtration studies showed that, at saturation, 3.5 moles of [14C]fructose 1,6-bisphosphate bound per mole of enzyme, with a dissociation constant of 0.06 mm determined by ultrafiltration. Inhibition by fructose 1,6-bisphosphate was greatly reduced by high pH, high ionic strength, or 0.2 m guanidine hydrochloride, some of which also stimulated enzyme activity. Under such conditions, [14C]fructose 1,6-bisphosphate still bound to the enzyme. A genetically desensitized glycerol kinase was purified to apparent homogeneity. It had a specific activity twice that of the normal enzyme, but was identical in size, charge, and subunit composition. Although the mutant enzyme still bound [14C]fructose 1,6-bisphosphate, it was totally insensitive to inhibition. The mutant enzyme also displayed a nonhyperbolic response to MgATP. Both the normal and mutant glycerol kinases were markedly stabilized against heat inactivation by glycerol, but not by fructose 1,6-bisphosphate. In the absence of specific ligands, about 12 sulfhydryl groups per molecule of enzyme reacted with 5,5'-dithiobis(nitro-2-benzoic acid). In the presence of glycerol, only two sulfhydryl groups per molecule reacted with the reagent, while fructose 1,6-bisphosphate had no effect. These results are discussed in terms of the subunit structure of the enzyme and in terms of the mechanism of fructose 1,6-bisphosphate inhibition of glycerol kinase.