Closed structure of phosphoglycerate kinase from Thermotoga maritima reveals the catalytic mechanism and determinants of thermal stability

The gene encoding the phosphoglycerate kinase (PGK) from the Antarctic Pseudomonas sp. TACII18 has been cloned and found to be inserted between the genes encoding for glyceraldhyde-3-phosphate dehydrogenase and fructose aldolase. The His-tagged and the native recombinant PGK from the psychrophilic Pseudomonas were expressed in Escherichia coli. The wild-type and the native recombinant enzymes displayed identical properties, such as a decreased thermostability and a 2-fold higher catalytic efficiency at 25°C when compared with the mesophilic PGK from yeast. These properties, which reflect typical features of cold-adapted enzymes, were strongly altered in the His-tagged recombinant PGK. The structural model of the psychrophilic PGK indicated that a key determinant of its low stability is the reduced number of salt bridges, surface charges, and aromatic interactions when compared with mesophilic and thermophilic PGK. Differential scanning calorimetry of the psychrophilic PGK revealed unusual variations in its conformational stability for the free and substrate-bound forms. In the free form, a heatlabile and a thermostable domain unfold independently. It is proposed that the heat-labile domain acts as a destabilizing domain, providing the required flexibility around the active site for catalysis at low temperatures.

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Bentahir

Feller

Aittaleb

et al. 2000

“…Therefore, significant "hinge bending" was suggested to be required for catalysis to occur (6). A major step forward in the understanding of catalysis by PGK was the determination of partially (10,11) and fully closed (12) crystal structures of the enzyme. The latter, in complex with transition state analogues (TSAs), defined for the first time the state of PGK responsible for its catalytic activity, in particular demonstrating that a catalytic triad of positively charged residues surrounds the transferring phosphoryl group.…”

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confidence: 99%

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

Zerrad

Merli

Schröder

et al. 2011

105

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 ...

“…The C-terminal domain is the binding site for ATP/ADP. The enzyme has a number of crystal structures (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). In many the two domains are in an open conformation with the substrates ϳ11 Å apart, which is much too far for direct phosphotransfer.…”

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confidence: 99%

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

Jakeman¹,

Ivory²,

Blackburn³

et al. 2003

We have previously reported dissociation constants for a range of bisphosphonate analogs of 1,3-bisphospho-D-glyceric acid binding to yeast phosphoglycerate kinase. Data for the unsymmetrical analogs were difficult to interpret because it was not clear in which of the two possible orientations these ligands bound. Here we report a novel NMR method for quantifying orientation preference based on relaxation effects induced by titration with CrADP, which is applied to these ligands. It is shown that all ligands can bind in both orientations but that the driving force for the orientational preference is to put the ␣,␣-difluoromethanephosphonate group in the "basic patch" (nontransferable phosphate) position. The relevance to the design of phosphoglycerate kinase inhibitors is discussed.Phosphoglycerate kinase (PGK) 1 (EC 2.7.2.3) is a glycolytic enzyme that catalyzes the following reaction, where MgADP and MgATP represent the magnesium complexes of ADP and ATP, respectively.