Phosphoglycerate kinase (PGK) is the enzyme responsible for the first ATP-generating step of glycolysis and has been implicated extensively in oncogenesis and its development. Solution small angle x-ray scattering (SAXS) data, in combination with crystal structures of the enzyme in complex with substrate and product analogues, reveal a new conformation for the resting state of the enzyme and demonstrate the role of substrate binding in the preparation of the enzyme for domain closure. Comparison of the x-ray scattering curves of the enzyme in different states with crystal structures has allowed the complete reaction cycle to be resolved both structurally and temporally. The enzyme appears to spend most of its time in a fully open conformation with short periods of closure and catalysis, thereby allowing the rapid diffusion of substrates and products in and out of the binding sites. Analysis of the open apoenzyme structure, defined through deformable elastic network refinement against the SAXS data, suggests that interactions in a mostly buried hydrophobic region may favor the open conformation. This patch is exposed on domain closure, making the open conformation more thermodynamically stable. Ionic interactions act to maintain the closed conformation to allow catalysis. The short time PGK spends in the closed conformation and its strong tendency to rest in an open conformation imply a springloaded release mechanism to regulate domain movement, catalysis, and efficient product release. Phosphoglycerate kinase (PGK)2 catalyzes the transfer of phosphate from 1,3-bisphosphoglycerate (1,3BPG) to ADP in the first ATP-generating step of the glycolytic pathway. As a major controller of flux through the pathway, PGK is a viable target for drugs against anaerobic pathogens, such as Trypanosoma and Plasmodium species, which depend solely on glycolysis for energy metabolism (1). In addition to its metabolic role, the phosphoryl transfer activity of PGK is important in the processing of antiretroviral prodrugs that take the form of L-nucleoside analogues (2). The rate-limiting step in the in vivo activation of such compounds has been demonstrated to be the addition of a third phosphate by PGK (3). A third activity of PGK is as a signaling molecule in chordates. It is integral in the response to hypoxia, when it is secreted from the cell and inhibits angiogenesis through a disulfide reductase activity that activates plasminogen autoproteolytic activity, producing angiostatin (4). This activity apparently uses the same mechanism as the normal metabolic reaction and can be inhibited competitively by 3-phosphoglycerate (3PG) or ADP (5). Consequently, PGK has a crucial role in oncogenesis and its development.PGK is composed of two similarly sized domains, both with Rossmann fold topology, termed the N-terminal domain, which binds the phosphoglycerate species 3PG and 1,3BPG, and the C-terminal domain, which binds the nucleotides ADP and ATP. In early crystal structures of PGK (6 -9), it was apparent that this state of the enzyme ...

Closure of the two domains of 3-phosphoglycerate kinase, upon substrate binding, is essential for the enzyme function. The available crystal structures cannot provide sufficient information about the mechanism of substrate assisted domain closure and about the requirement of only one or both substrates, since lattice forces may hinder the large scale domain movements. In this study the known X-ray data, obtained for the open and closed conformations, were probed by solution small-angle Xray scattering experiments. The results prove that binding of both substrates is essential for domain closure. Molecular graphical analysis, indeed, reveals formation of a double-sided H-bond network, which affects substantially the shape of the main molecular hinge at b-strand L, under the concerted action of both substrates.

The three-dimensional structure of the enzyme 3-isopropylmalate dehydrogenase from the bacterium Thermus thermophilus in complex with Mn 2+ , its substrate isopropylmalate and its co-factor product NADH at 2.0 A resolution features a fully closed conformation of the enzyme. Upon closure of the two domains, the substrate and the co-factor are brought into precise relative orientation and close proximity, with a distance between the C2 atom of the substrate and the C4N atom of the pyridine ring of the co-factor of approximately 3.0A. The structure further shows binding of a K + ion close to the active site, and provides an explanation for its known activating effect. Hence, this structure is an excellent mimic for the enzymatically competent complex. Using high-level QM/MM calculations, it may be demonstrated that, in the observed arrangement of the reactants, transfer of a hydride from the C2 atom of 3-isopropylmalate to the C4N atom of the pyridine ring of NAD + is easily possible, with an activation energy of approximately 15 kcalÁmol À1 . The activation energy increases by approximately 4-6 kcalÁmol À1 when the K + ion is omitted from the calculations. In the most plausible scenario, prior to hydride transfer the eamino group of Lys185 acts as a general base in the reaction, aiding the deprotonation reaction of 3-isopropylmalate prior to hydride transfer by employing a low-barrier proton shuttle mechanism involving a water molecule. DatabaseStructural data have been submitted to the Protein Data Bank under accession number 4F7I.Abbreviations IPMDH, 3-isopropylmalate dehydrogenase; IPM, 3-isopropylmalate; QM/MM, hybrid quantum mechanics molecular mechanics.