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Reactions of D-glyceraldehyde 3-phosphate dehydrogenase with chromophoric thiol reagents.

ABSTRACT: The kinetics of the reaction of d-glyceraldehyde 3-phosphate dehydrogenase with 5,5'-dithiobis-(2-nitrobenzoic acid) show that NAD(+) dissociates from the enzyme before the reaction. In contrast 2-chloromercuri-4-nitrophenol reacts with the holoenzyme without prior dissociation of NAD(+). These studies and observations on the dissociation constant of NAD(+) to the lobster enzyme show that NAD(+) must dissociate from sites modified by substrates during the reductive dephosphorylation of 1,3-diphosphoglycerate. All four sites per tetramer of the apoenzyme are acylated by 1,3-diphosphoglycerate. Hydrolysis of the acyl-enzyme occurs at a significant rate even in the absence of NAD(+), which may explain previous estimates that only two sites per tetramer can readily be acylated.

SUBMITTER: Harrigan PJ

PROVIDER: S-EPMC1177227 | biostudies-other | 1971 Sep

REPOSITORIES: biostudies-other

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A new class of chromophoric organomercurials and their reactions with D-glyceraldehyde 3-phosphate dehydrogenase.

Project description:THE SYNTHESES OF THE FOLLOWING ORGANOMERCURIALS ARE DESCRIBED: 2-chloromercuri-4-nitrophenol, 2-chloromercuri-4,6-dinitrophenol, 4-chloromercuri-2-nitrophenol and 2,6-dichloromercuri-4-nitrophenol. All four organomercurials show large spectral changes in the visible spectrum when thiols displace a more weakly bound ligand such as EDTA from the mercury atom. These spectral changes are primarily associated with pK perturbation of the nitrophenols. The mercurials are therefore chromophoric probes for thiol groups in proteins and other thiols of biological interest. The enzyme d-glyceraldehyde 3-phosphate dehydrogenase from lobster muscle is used as a model system in which the properties of the organomercurials may be illustrated. In particular it is shown how d-glyceraldehyde 3-phosphate dehydrogenase carboxymethylated at the active site may be mercurated at a specific site. This mercurial derivative may be crystallized and shown to be isomorphous with the parent enzyme. The mercurials also act as ;reporter groups' by monitoring phosphate or pyrophosphate binding to the enzyme. The mercurials may also be used to estimate cations by an EDTA displacement method.

| S-EPMC1185233 | biostudies-other

Direct and nitroxyl (HNO)-mediated reactions of acyloxy nitroso compounds with the thiol-containing proteins glyceraldehyde 3-phosphate dehydrogenase and alkyl hydroperoxide reductase subunit C.

Project description:Nitroxyl (HNO) reacts with thiols, and this reactivity requires the use of donors with 1-nitrosocyclohexyl acetate, pivalate, and trifluoroacetate, forming a new group. These acyloxy nitroso compounds inhibit glyceraldehyde 3-phosphate dehydrogenase (GAPDH) by forming a reduction reversible active site disulfide and a reduction irreversible sulfinic acid or sulfinamide modification at Cys244. Addition of these acyloxy nitroso compounds to AhpC C165S yields a sulfinic acid and sulfinamide modification. A potential mechanism for these transformations includes nucleophilic addition of the protein thiol to a nitroso compound to yield an N-hydroxysulfenamide, which reacts with thiol to give disulfide or rearranges to sulfinamides. Known HNO donors produce the unsubstituted protein sulfinamide as the major product, while the acetate and pivalate give substituted sulfinamides that hydrolyze to sulfinic acids. These results suggest that nitroso compounds form a general class of thiol-modifying compounds, allowing their further exploration.

| S-EPMC3798158 | biostudies-literature

Heme binding properties of glyceraldehyde-3-phosphate dehydrogenase.

Project description:Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that also functions in transcriptional regulation, oxidative stress, vesicular trafficking, and apoptosis. Because GAPDH is required for the insertion of cellular heme into inducible nitric oxide synthase [Chakravarti, R., et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 18004-18009], we extensively characterized the heme binding properties of GAPDH. Substoichiometric amounts of ferric heme bound to GAPDH (one heme per GAPDH tetramer) to form a low-spin complex with UV-visible maxima at 362, 418, and 537 nm and when reduced to ferrous gave maxima at 424, 527, and 559 nm. Ferric heme association and dissociation rate constants at 10 °C were as follows: k(on) = 17800 M(-1) s(-1), k(off1) = 7.0 × 10(-3) s(-1), and k(off2) = 3.3 × 10(-4) s(-1) (giving approximate affinities of 19-390 nM). Ferrous heme bound more poorly to GAPDH and dissociated with a k(off) of 4.2 × 10(-3) s(-1). Magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopic data on the ferric, ferrous, and ferrous-CO complexes of GAPDH showed that the heme is bis-ligated with His as the proximal ligand. The distal ligand in the ferric complex was not displaced by CN(-) or N(3)(-) but in the ferrous complex could be displaced by CO at a rate of 1.75 s(-1) (for >0.2 mM CO). Studies with heme analogues revealed selectivity toward the coordinating metal and porphyrin ring structure. The GAPDH-heme complex was isolated from bacteria induced to express rabbit GAPDH in the presence of ?-aminolevulinic acid. Our finding of heme binding to GAPDH expands the protein's potential roles. The strength, selectivity, reversibility, and redox sensitivity of heme binding to GAPDH are consistent with it performing heme sensing or heme chaperone-like functions in cells.

| S-EPMC3549054 | biostudies-literature

Testis-specific glyceraldehyde-3-phosphate dehydrogenase: origin and evolution.

Project description:BACKGROUND: Glyceraldehyde-3-phosphate dehydrogenase (GAPD) catalyses one of the glycolytic reactions and is also involved in a number of non-glycolytic processes, such as endocytosis, DNA excision repair, and induction of apoptosis. Mammals are known to possess two homologous GAPD isoenzymes: GAPD-1, a well-studied protein found in all somatic cells, and GAPD-2, which is expressed solely in testis. GAPD-2 supplies energy required for the movement of spermatozoa and is tightly bound to the sperm tail cytoskeleton by the additional N-terminal proline-rich domain absent in GAPD-1. In this study we investigate the evolutionary history of GAPD and gain some insights into specialization of GAPD-2 as a testis-specific protein. RESULTS: A dataset of GAPD sequences was assembled from public databases and used for phylogeny reconstruction by means of the Bayesian method. Since resolution in some clades of the obtained tree was too low, syntenic analysis was carried out to define the evolutionary history of GAPD more precisely. The performed selection tests showed that selective pressure varies across lineages and isoenzymes, as well as across different regions of the same sequences. CONCLUSIONS: The obtained results suggest that GAPD-1 and GAPD-2 emerged after duplication during the early evolution of chordates. GAPD-2 was subsequently lost by most lineages except lizards, mammals, as well as cartilaginous and bony fishes. In reptilians and mammals, GAPD-2 specialized to a testis-specific protein and acquired the novel N-terminal proline-rich domain anchoring the protein in the sperm tail cytoskeleton. This domain is likely to have originated by exonization of a microsatellite genomic region. Recognition of the proline-rich domain by cytoskeletal proteins seems to be unspecific. Besides testis, GAPD-2 of lizards was also found in some regenerating tissues, but it lacks the proline-rich domain due to tissue-specific alternative splicing.

| S-EPMC3224139 | biostudies-literature

Analysis of transcription profile of the plastidial glyceraldehyde-3-phosphate dehydrogenase mutants

Project description:Aerial parts (AP) and roots of wild-type plants were compared with plastidial glyceraldehyde-3-phosphate dehydrogenase double mutants (gapcp1gapcp2). These mutants were also compared with conditional mutants after GAPCp induction.

2015-10-30 | E-MTAB-3262 | biostudies-arrayexpress

Reactions of glyceraldehyde 3-phosphate dehydrogenase sulfhydryl groups with bis-electrophiles produce DNA-protein cross-links but not mutations.

Project description:The environmental contaminant 1,2-dibromoethane and diepoxybutane, an oxidation product of the important industrial chemical butadiene, are bis-functional electrophiles and are known to be mutagenic and carcinogenic. One mechanism by which bis-electrophiles can exert their toxic effects is through the induction of genotoxic and mutagenic DNA-peptide cross-links. This mechanism has been shown in systems overexpressing the DNA repair protein O6 -alkylguanine DNA-alkyltransferase (AGT) or glutathione S-transferase and involves reactions with nucleophilic cysteine residues. The hypothesis that DNA-protein cross-link formation is a more general mechanism for genotoxicity by bis-electrophiles was investigated by screening nuclear proteins for reactivity with model monofunctional electrophiles. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was identified as a candidate because of the nucleophilicity of two cysteine residues (Cys152 and Cys246) in reaction screens with model electrophiles (Dennehy, M. K. et al. (2006) Chem. Res. Toxicol. 19, 20-29). Incubation of GAPDH with bis-electrophiles resulted in inhibition of its catalytic activity, but only at high concentrations of diepoxybutane. In vitro assays indicated DNA-GAPDH cross-link formation in the presence of diepoxybutane, and bis-electrophile reactivity at Cys246 was confirmed using mass spectral analysis. In contrast to AGT, overexpression of human GAPDH in Escherichia coli did not enhance mutagenesis by diepoxybutane. We propose that the lack of mutational enhancement is in part due to the inherently lower reactivity of GAPDH toward bis-electrophiles as well as the reduced DNA binding ability relative to AGT, preventing the in vivo formation of DNA-protein cross-links and enhanced mutagenesis.

| S-EPMC4049368 | biostudies-literature

The active chemical state of D-glyceraldehyde 3-phosphate in its reactions with D-glyceraldehyde 3-phosphate dehydrogenase, aldolase and triose phosphate isomerase.

Project description:Glyceraldehyde 3-phosphate exists as the geminal diol and the free aldehyde in the molar ratio 29:1 in aqueous solution. The rate constant of the conversion of diol into aldehyde is 8.7x10(-2)sec.(-1) in the pH range 7.3-8.6 at 20 degrees . The free aldehyde is the substrate for d-glyceraldehyde 3-phosphate dehydrogenase. Over a wide concentration range of enzyme the rate of conversion of diol into aldehyde is the rate-limiting process in the catalytic oxidation of d-glyceraldehyde 3-phosphate by NAD(+). Aldolase and triose phosphate isomerase both liberate d-glyceraldehyde 3-phosphate as the aldehyde. This suggests that the relatively slow diol-aldehyde interconversion does not restrict the rate of glycolysis.

| S-EPMC1184790 | biostudies-other

Structure of apo-glyceraldehyde-3-phosphate dehydrogenase from Synechococcus PCC7942.

Project description:The crystal structure of NADP-dependent apo-glyceraldehyde-3-phosphate dehydrogenase (apo-GAPDH) from Synechococcus PCC 7942 is reported. The crystal structure was solved by molecular replacement and refined to an R of 21.7% and R(free) of 27.5% at 2.9 angstroms resolution. The structural features of apo-GAPDH are as follows. The S-loop has an extremely flexible conformation and the sulfate ion is only taken into the classical P(i) site. A structural comparison with holo-GAPDHs indicated that the S-loop fixation is essential in the discrimination of NADP and NAD molecules.

| S-EPMC2242934 | biostudies-literature

Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase Is Phosphorylated during Seed Development.

Project description:Cytosolic glyceraldehyde-3-phosphate dehydrogenase (NAD-GAPDH) is involved in a critical energetic step of glycolysis and also has many important functions besides its enzymatic activity. The recombinant wheat NAD-GAPDH was phosphorylated in vitro at Ser205 by a SNF1-Related protein kinase 1 (SnRK1) from wheat heterotrophic (but not from photosynthetic) tissues. The S205D mutant enzyme (mimicking the phosphorylated form) exhibited a significant decrease in activity but similar affinity toward substrates. Immunodetection and activity assays showed that NAD-GAPDH is phosphorylated in vivo, the enzyme depicting different activity, abundance and phosphorylation profiles during development of seeds that mainly accumulate starch (wheat) or lipids (castor oil seed). NAD-GAPDH activity gradually increases along wheat seed development, but protein levels and phosphorylation status exhibited slight changes. Conversely, in castor oil seed, the activity slightly increased and total protein levels do not significantly change in the first half of seed development but both abruptly decreased in the second part of development, when triacylglycerol synthesis and storage begin. Interestingly, phospho-NAD-GAPDH levels reached a maximum when the seed switch their metabolism to mainly support synthesis and accumulation of carbon reserves. After this point the castor oil seed NAD-GAPDH protein levels and activity highly decreased, and the protein stability assays showed that the protein would be degraded by the proteasome. The results presented herein suggest that phosphorylation of NAD-GAPDH during seed development would have impact on the partitioning of triose-phosphate between different metabolic pathways and cell compartments to support the specific carbon, energy and reducing equivalent demands during synthesis of storage products.

| S-EPMC5387080 | biostudies-literature

A cryoprotectant induces conformational change in glyceraldehyde-3-phosphate dehydrogenase.

Project description:Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, catalyses the conversion of D-glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate. While mammalian and yeast GAPDHs are multifunctional proteins that have additional functions beyond those involved in glycolysis, including reactions related to nuclear RNA transport, DNA replication/repair, membrane fusion and cellular apoptosis, Escherichia coli GAPDH (ecGAPDH) has only been reported to function in glycolysis. The S-loop of GAPDH is required for interaction with its cofactor and with other proteins. In this study, the three-dimensional crystal structure of GAPDH treated with trehalose is reported at 2.0?Å resolution. Trehalose was used as a cryoprotectant for the GAPDH crystals. The structure of trehalose-bound ecGAPDH was compared with the structures of both NAD+-free and NAD+-bound ecGAPDH. At the S-loop, the bound trehalose in the GAPDH structure induces a 2.4° rotation compared with the NAD+-free ecGAPDH structure and a 3.1° rotation compared with the NAD+-bound ecGAPDH structure.

| S-EPMC5931139 | biostudies-literature

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