Anion Activation of 3-Phosphoglycerate Kinase Requires Domain Closure

Abstract

3-Phosphoglycerate kinase is a 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 effectors, 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.

Structural, Kinetic, and Calorimetric Characterization of the Cold-active Phosphoglycerate Kinase from the AntarcticPseudomonas sp. TACII18

“…Unlike the mesophilic PGK from yeast which shows a neutral pI (28) and an optimal pH for activity at pH 8.0, the pI of the psychrophilic enzyme is acidic, pI 4.9, and it is active over a broad pH range (6.5-10), with an optimal activity at pH 8.0 (data not shown). The cold-adapted enzyme is activated by sulfate ions at concentrations below 20 mM and is inhibited at concentrations above 50 mM, as already reported for mesophilic PGK (29,30). The first 15 amino acids of the N terminus were determined by Edman degradation and were found to be similar to that deduced from the gene sequence, displaying a removed formylmethionine and sharing substantial homology with other PGK sequences from different sources.…”

Stability Domains, Substrate-induced Conformational Changes, and Hinge-bending Motions in a Psychrophilic Phosphoglycerate Kinase

“…The lack of ͚⌬H cal difference between the free and closed forms of PsPGK (TABLE ONE) also supports this view. Finally, in a thoughtful study of PGK activation, the authors concluded that it cannot be decided whether anion activation is a consequence of domain closure or anion binding itself mediates the closure by connecting the two domains (23). From our results, it seems that the latter hypothesis is valid, as demonstrated by the apparent domain closure induced by the sulfate anion activator (Fig.…”

“…These results clearly demonstrate that the Mg 2ϩ -binding site belongs to the heat-stable DSC transition. Finally, the sulfate ion can be viewed as a test case for our assignment of the DSC transitions because it has been reported to bind both to the N-(Arg 61 ) and C-(Lys 192 ) domains (23). And indeed, increasing Na 2 SO 4 concentrations affect both the heat-labile and heat-stable DSC transitions (Fig.…”

Orientation of 1,3-Bisphosphoglycerate Analogs Bound to Phosphoglycerate Kinase

“…has occurred as to whether 1,3-BPG and its analogs bind at more than one site on PGK (16), and it is now reasonably clear that at least in the open form it only has a single major binding site (13). Finally, by using a low concentration of both enzyme and CrADP along with tight binding PGK ligands we can ensure that essentially all of the paramagnetic relaxation of the ligand occurs while bound to the enzyme, and no significant relaxation occurs in a CrADP⅐ligand binary complex.…”

“…Another subject of intense study is the kinetics of the reaction, which is activated by anions (15). Although the issue has not been resolved, it is likely that an anion binding site is formed when the two domains close (16).…”