Synergistic effects of substrate-induced conformational changes in phosphoglycerate kinase activation

Abstract

Phosphoglycerate kinase (PGK), a key enzyme in glycolysis, catalyses the transfer of a phosphoryl-group from 1,3-bisphosphoglycerate to ADP to form 3-phosphoglycerate and ATP. Despite extensive kinetic and structural investigations over more than two decades, the conformation assumed by this enzyme during catalysis remained unknown. Here we present the 2.8 A crystal structure of a ternary complex of PGK from Trypanosoma brucei, the causative agent of sleeping sickness. This structure determination relied on a procedure in which fragments containing less than 10% of the scattering mass were successively positioned in the unit cell to obtain phases. The PGK ternary complex exhibits a dramatic closing of the large cleft between the two domains seen in all previous studies, thereby bringing the two ligands, 3-phosphoglycerate and ADP into close proximity. Our results demonstrate that PGK is a hinge-bending enzyme, reveal a novel mechanism in which substrate-induced effects combine synergistically to induce major conformational changes and, to our knowledge, afford the first observation of the PGK active site in a catalytic conformation.

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

“…As shown by crystallographic studies, the triose substrate binds to a basic patch in the N-terminal domain (9) whereas the nucleotide substrate binds to the C-terminal domain (10). Six three-dimensional structures have been solved by x-ray crystallography: the PGK from horse muscle (11)(12)(13), yeast (14,15), pig muscle (9,16), Bacillus stearothermophilus (10), Trypanosoma brucei (17), and Thermotoga maritima (18). Until recently, the reported structures were in a conformational state incompatible with substrate catalysis.…”

“…Until recently, the reported structures were in a conformational state incompatible with substrate catalysis. The T. brucei ternary complex has been crystallized in the catalytically competent conformation, however, and has shown that the substrates are close to each other and well aligned for phosphoryl transfer (17). Interestingly, the PGK complete hinge closure occurs only when both substrates are bound and depends on their synergistically induced conformational changes.…”

Crowding and function reunite

“…One substrate, ADP, binds an inner face, whereas the other substrate, 1,3-bisphosphoglycerate, binds the opposing inner face. Bringing the lobes together is thought to be involved in catalysis (8,9). As such, crowding would be predicted to increase the enzyme's activity.…”

Structure, function, and folding of phosphoglycerate kinase are strongly perturbed by macromolecular crowding

“…In the crystal structure, the ADP and diphosphoglycerate binding sites, each located at an N and C subunit, are separated. It has been suggested that a large-scale conformational change (2) is necessary to bring the two subunits together when the phosphoryl group is catalytically transferred, and a hinge-bending mechanism has been postulated (3), bringing together both substrates at the inner surfaces of the C and N subdomains (4,5).…”