Anion binding sites in the active center of d-glyceraldehyde-3-phosphate dehydrogenase

doi:10.1016/S0022-2836(76)80083-6

Journal of Molecular Biology

Volume 107, Issue 4, 15 November 1976, Pages 571-576

https://doi.org/10.1016/S0022-2836(76)80083-6 Get rights and content

Crystals of lobster holo-d-glyceraldehyde-3-phosphate dehydrogenase, grown in ammonium sulfate, were dialyzed against sodium citrate buffer at pH 6·2. This procedure caused two sulfate ions in each active center to be displaced by one bridging citrate ion. The position of these two ion sites can be associated with the terminal phosphate group of the substrate and the participating inorganic phosphate. The degree of occupancy by the sulfate ions is the same in all four subunits within experimental error.

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Structure of glyceraldehyde-3-phosphate dehydrogenase from Paracoccidioides lutzii in complex with an aldonic sugar acid
2024, Biochimie
The pathogen Paracoccidioides lutzii (Pb01) is found in South America countries Colombia, Ecuador, Venezuela and Brazil, especially in the central, west, and north regions of the latter. It belongs to the Ajellomycetaceae family, Onygenales order, and is typically thermodimorphic, presenting yeast cells when it grows in animal tissues, but mycelia when in the environment, where it produces the infectious propagule. This fungus is one of the etiologic agents of Paracoccidioidomycosis (PCM), the most important endemic fungal infection in Latin America. Investigations on its genome have contributed to a better understanding about its metabolism and revealed the complexity of several metabolic glycolytic pathways. Glyceraldehyde-3-Phosphate Dehydrogenase from Paracoccidioides lutzii (PlGAPDH) is considered a moonlighting protein and participates in several biological processes of this pathogen. The enzyme was expressed and purified, as seen in SDS-PAGE gel, crystallized and had its three dimensional structure (3D) determined in complex with NAD⁺, a sulphate ion and d-galactonic acid, therefore, a type of ‘GAA site’. It is the first GAPDH structure to show this chemical type in this site and how this protein can bind an acid derived from oxidation of a linear hexose.
Glyceraldehyde-3-phosphate dehydrogenase from Sulfolobus solfataricus
2001, Methods in Enzymology
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the oxidative phosphorylation of o-glyceraldehyde 3-phosphate to form 1,3-diphosphoglycerate and is found in both glycolytic and gluconeogenic metabolic pathways. The reaction mechanism of the bacterial and eukaryotic enzyme has been studied in detail and proceeds through a thioester-acyl enzyme intermediate with a cysteine amino acid in the enzyme active site. The S. solfataricus enzyme has relatively high sequence identity (45-50%) to other archaeal GAPDHs. The low sequence similarity between archaeal GAPDHs and enzymes from the two other kingdoms, as well as the difficulty in aligning residues implicated in the catalytic mechanism, have led to the suggestion that archaeal GAPDHs are unrelated to their bacterial and eukaryotic counterparts and show a convergent molecular evolution in the catalytic region of their structure.
Crystal structure of the glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon sulfolobus solfataricus
1999, Journal of Molecular Biology
The enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the archaea shows low sequence identity (16–20 %) with its eubacterial and eukaryotic counterparts. The crystal structure of the apo GAPDH from Sulfolobus solfataricus has been determined by multiple isomorphous replacement at 2.05 Å resolution. The enzyme has several differences in secondary structure when compared with eubacterial GAPDHs, with an overall increase in the number of α-helices. There is a relocation of the active-site residues within the catalytic domain of the enzyme. The thermostability of the S. solfataricus enzyme can be attributed to a combination of an ion pair cluster and an intrasubunit disulphide bond.
Kinetic evidence for a reversible isomerization of pig muscle glyceraldehyde-3-phosphate dehydrogenase in its crystallization medium
1988, Archives of Biochemistry and Biophysics
Ammonium sulfate, a typical component of crystallization media of proteins, stabilizes an inactive conformation of pig muscle glyceraldehyde-3-phosphate dehydrogenase. In fact, in the presence of ammonium sulfate the reconstitution of the catalytically active holoenzyme from the apoenzyme and NAD is not instantaneous, as in the case of enzymes from Bacillus stearothermophilus and the Mediterranean lobster Palinurus vulgaris. With pig muscle enzyme, at pH 6.0, the time course of formation of the characteristic Racker band can be monitored by a rapid mixing stopped flow technique. Activation follows a single exponential curve with a rate constant independent of the concentration of both NAD and protein and, therefore, appears to be limited by a slow protein isomerization (k = 7 ± 2 s⁻¹). Accordingly, when the apoenzyme is simultaneously exposed to NAD and either glyceraldehyde 3-phosphate or 1,3-bisphosphoglycerate, the ensuing reactions (the redox and the acylation steps, respectively) are kinetically limited by the same protein isomerization. At pH 7.0 and 8.0, however, two among the four active sites react with NAD at an unmeasurably high rate, while the other two sites behave as they do at pH 6.O. When the pig muscle apoenzyme is preincubated and allowed to react with either glyceraldehyde 3-phosphate or 1,3-bisphosphoglycerate before the rapid mixing with NAD, both the redox reaction and the NAD-dependent activation of apo-acyl-enzyme toward arsenolysis become unmeasurably fast. Similarly, when the sulfate in the medium is replaced by ions such as phosphate and citrate, the reconstitution of the active holoenzyme is practically instantaneous. Thus, the slow protein isomerization observed in the presence of sulfate and abolished by competing substrates and anions is diagnostic of a structural state of the pig muscle apoenzyme, which is induced by sulfate ions bound within the enzyme active site.
Microenvironment of the enzyme-bound NADH is different in lobster and pig muscle glyceraldehyde-3-phosphate dehydrogenase microcrystals
1986, Archives of Biochemistry and Biophysics
Two possible consequences of crystal lattice formation were studied with glyceraldehyde-3-phosphate dehydrogenases isolated from lobster (Palinurus vulgaris) and pig muscle: (i) changes in the microenvironment of the NADH-binding site as detected by fluorescence polarization, and (ii) differences in the maximal activities of the microcrystalline enzymes as compared to those in solution, (i) In solution practically no difference was found between the polarization values of the enzyme-NADH and the catalytic intermediate 3-phosphoglyceroyl-enzyme-NADH complexes whether with lobster or with pig enzyme. In microcrystalline state a similar effect was found with the lobster enzyme. However, fluorescence polarization of NADH bound to the pig enzyme was significantly different in the presence and in the absence of the 3-phosphoglyceroyl group. This indicates some change in the microenvironment of the pig enzyme-bound NADH which occurs upon decomposition of the catalytic intermediate. (ii) The difference between the microcrystalline lobster and pig muscle glyceraldehyde-3-phosphate dehydrogenases pertains also to their functional properties. Packing of soluble pig muscle enzyme into a crystal lattice stabilizes a unique protein conformation of extremely low activity (about 3% of that measured in solution). The maximal molar activity of the lobster enzyme is identical in crystalline state and in solution, which is an exceptional phenomenon.
Visualization of electrostatic recognition by enzymes for their ligands and cofactors
1985, Journal of Molecular Graphics
Electrostatic potentials for several enzymes and their guest molecules such as ligands and/or cofactors are individually calculated and illustrated as colour codes on their tertiary structures using a raster graphics technique. Two kinds of potential surface were illustrated on the molecular surface of the guest molecule. One is due to the host enzyme (H-on-G potential surface) and the other is due to the guest molecule itself (G-on-G potential surface). Remarkable mutual complementarities between the H-on-G and G-on-G potential surfaces were found in most cases. These give a semi-quantitative indication of the important role of electrostatic recognition in enzymes.

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^†: Present address: Department of Biochemistry, University of Mississippi Medical Center, 2500 North State Street, Jackson, Miss. 39212, U.S.A.

^‡: Present address: Biochemistry Department, Indiana University Medical Center, Indianapolis, Ind. 46207, U.S.A.

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Journal of Molecular Biology

Anion binding sites in the active center of d-glyceraldehyde-3-phosphate dehydrogenase

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Biol. Chem.

J. Mol. Biol.

Methods Enzymol.

J. Appl. Crystallogr.

Structure of glyceraldehyde-3-phosphate dehydrogenase from Paracoccidioides lutzii in complex with an aldonic sugar acid

Glyceraldehyde-3-phosphate dehydrogenase from Sulfolobus solfataricus

Crystal structure of the glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon sulfolobus solfataricus

Kinetic evidence for a reversible isomerization of pig muscle glyceraldehyde-3-phosphate dehydrogenase in its crystallization medium

Microenvironment of the enzyme-bound NADH is different in lobster and pig muscle glyceraldehyde-3-phosphate dehydrogenase microcrystals

Visualization of electrostatic recognition by enzymes for their ligands and cofactors