A Spring-loaded Release Mechanism Regulates Domain Movement and Catalysis in Phosphoglycerate Kinase

Abstract

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 ...

Inducing phase changes in crystals of macromolecules: Status and perspectives for controlled crystal dehydration

“…alaska) chloroplast photosystem I (PSI) were kindly provided by Prof. Nathan Nelson (Department of Molecular Biology and Biochemistry, Tel Aviv University, Israel). Crystals of human phosphoglycerate kinase (hsPGK) were grown as previously described Zerrad et al, 2011). Crystals of L. lactis b-phosphoglucomutase (bPGM) were grown as described .…”

Interaction of Human 3-Phosphoglycerate Kinase with Its Two Substrates: Is Substrate Antagonism a Kinetic Advantage?

“…It has been demonstrated that the enzyme remains mostly in an open conformation, with short bursts of closure for catalysis. 17 In this situation, an enhanced mechanism for substrate and product exchange has clear advantages.…”

“…For phosphotransfer to be allowed, upon the binding of both substrates, an extensive hinge bending motion occurs that leads to a "closed" conformation, resulting in amalgamation of the sites. 16,17 While extensive structural studies have been carried out on the open form of PGKs from a number of sources (Kovári and Vas 18 and references cited therein), the first structure of a closed form was, until recently (see the text below), obtained only for the enzymes from Trypanosoma brucei (the causative agent of sleeping sickness) 19 and Thermotoga maritima. 20 In these works, it was shown that, in the ternary PGK·PG·ADP complex, there was a dramatic closing of the large cleft that separates the two substrate sites, thereby bringing bound PG and ADP in close proximity.…”

Thioredoxin-dependent Redox Regulation of Chloroplastic Phosphoglycerate Kinase from Chlamydomonas reinhardtii

“…This analysis was performed using the model of the reduced protein and imposing the formation of the disulfide bond between Cys 227 and Cys 361 by energy minimization to adjust the protein conformation. It has been demonstrated for human PGK that after binding of the two substrates, the enzyme assumes a half-closed conformation in which the two substrates appear too distant from each other to allow phosphoryl transfer (28). A significant "hinge bending" movement, which leads the enzyme to a closed conformation, was suggested to allow enzyme catalysis to occur.…”

“…The N-terminal domain contains the 3-PGA binding site, whereas the ATP cofactor binds to the C-terminal domain. After substrate binding, a conformational change brings the two domains in close proximity, generating the so-called "closed conformation" that ensures catalytic reactions (28).…”