Anion activation of 3-phosphoglycerate kinase requires domain closure

3-Phosphoglycerate kinase isa typical two-domain "hinge-bending" enzyme, which is known to be regulated by multivalent anions. Here a relationship between this regulation and the hinge-bending domain closure is proposed on the basis of enzyme kinetic analysis and molecular modeling. Activation of the pig muscle enzyme at low concentrations and inhibition at high concentrations of various anionic analogues of the substrate 3-phosphoglycerate or of the nonsubstrate metal-free ATP are described by a two-site model assuming separate sites for activation and inhibition, respectively. Kinetic experiments with various pairs of analogues suggest the presence of a common site for activation by all effecters, separate from the catalytic site for 3-phosphoglycerate; and a common site for inhibition, except for metal-free ATP, identical with the catalytic site of 3-phosphoglycerate. An additional inhibiting site for all of the anions investigated, including metal-free ATP, is also proposed. A similar two-site model can describe activation of the enzyme by a large excess of each substrate; :here the ligand binds to the catalytic site as a substrate and to the regulatory site as an activator. Activation is exerted not only by the physiological substrate, 3-phophoglycerate, but also by a synthetic weak substrate. The activity in the reaction with 3-phosphoglycerate and MgATP is greatly enhanced by the simultaneous presence of the weak substrate. This finding clearly proves the existence of a regulatory site, separate from the catalytic site. This regulatory site, however, may only exist in the catalytically competent closed conformation of the enzyme, as indicated by molecular modeling. Docking of the regulator anions into the known X-ray structures of the enzyme revealed the appearance of an anion binding site between the two domains, including the invariant residues of Lys-215 (C-domain) and of Arg-65 among other residues of the basic cluster (N-domain), as a consequence of the large-scale substrate-induced conformational change that leads to domain closure.