Yeast Glyceraldehyde 3-phosphate Dehydrogenase Research Articles

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

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.

Glyceraldehyde-3-phosphate dehydrogenase is largely unresponsive to low regulatory levels of hydrogen peroxide in Saccharomyces cerevisiae

BackgroundThe reversible oxidation of protein SH groups has been considered to be the basis of redox regulation by which changes in hydrogen peroxide (H2O2) concentrations may control protein function. Several proteins become S-glutathionylated following exposure to H2O2 in a variety of cellular systems. In yeast, when using a high initial H2O2 dose, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as the major target of S-glutathionylation which leads to reversible inactivation of the enzyme. GAPDH inactivation by H2O2 functions to reroute carbohydrate flux to produce NADPH. Here we report the effect of low regulatory H2O2 doses on GAPDH activity and expression in Saccharomyces cerevisiae.ResultsA calibrated and controlled method of H2O2 delivery - the steady-state titration - in which cells are exposed to constant, low, and known H2O2 concentrations, was used in this study. This technique, contrary to the common bolus addition, allows determining which H2O2 concentrations trigger specific biological responses. This work shows that both in exponential- and stationary-phase cells, low regulatory H2O2 concentrations induce a large upregulation of catalase, a fingerprint of the cellular oxidative stress response, but GAPDH oxidation and the ensuing activity decrease are only observed at death-inducing high H2O2 doses. GAPDH activity is constant upon incubation with sub-lethal H2O2 doses, but in stationary-phase cells there is a differential response in the expression of the three GAPDH isoenzymes: Tdh1p is strongly upregulated while Tdh2p/Tdh3p are slightly downregulated.ConclusionsIn yeast GAPDH activity is largely unresponsive to low to moderate H2O2 doses. This points to a scenario where (a) cellular redoxins efficiently cope with levels of GAPDH oxidation induced by a vast range of sub-lethal H2O2 concentrations, (b) inactivation of GAPDH cannot be considered a sensitive biomarker of H2O2-induced oxidation in vivo. Since GAPDH inactivation only occurs at cell death-inducing high H2O2 doses, GAPDH-dependent rerouting of carbohydrate flux is probably important merely in pathophysiological situations. This work highlights the importance of studying H2O2-induced oxidative stress using concentrations closer to the physiological for determining the importance of protein oxidation phenomena in the regulation of cellular metabolism.

Tomato bushy stunt virus Co-Opts the RNA-Binding Function of a Host Metabolic Enzyme for Viral Genomic RNA Synthesis

Tomato bushy stunt virus (TBSV), a plus-stranded [(+)] RNA plant virus, incorporates the host metabolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) into the viral replicase complex. Here, we show that, during TBSV replication in yeast, the yeast GAPDH Tdh2p moves from the cytosol to the peroxisomal membrane surface, the site of viral RNA synthesis. In yeast cells lacking Tdh2p, decreasing the levels of its functionally redundant homolog Tdh3p inhibited TBSV replication and resulted in equivalent levels of (+) and minus-stranded [(-)] viral RNA, in contrast to the hallmark excess of (+)RNA. Tdh2p specifically bound an AU pentamer sequence in the (-)RNA, suggesting that GAPDH promotes asymmetric RNA synthesis by selectively retaining the (-)RNA template in the replicase complex. Downregulation of GAPDH in a natural plant host decreased TBSV genomic RNA accumulation. Thus, TBSV co-opts the RNA-binding function of a metabolic protein, helping convert the host cell into a viral factory.

NO-mediated apoptosis in yeast

Nitric oxide (NO) is a small molecule with distinct roles in diverse physiological functions in biological systems, among them the control of the apoptotic signalling cascade. By combining proteomic, genetic and biochemical approaches we demonstrate that NO and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are crucial mediators of yeast apoptosis. Using indirect methodologies and a NO-selective electrode, we present results showing that H2O2-induced apoptotic cells synthesize NO that is associated to a nitric oxide synthase (NOS)-like activity as demonstrated by the use of a classical NOS kit assay. Additionally, our results show that yeast GAPDH is a target of extensive proteolysis upon H2O2-induced apoptosis and undergoes S-nitrosation. Blockage of NO synthesis with Nomega-nitro-L-arginine methyl ester leads to a decrease of GAPDH S-nitrosation and of intracellular reactive oxygen species (ROS) accumulation, increasing survival. These results indicate that NO signalling and GAPDH S-nitrosation are linked with H2O2-induced apoptotic cell death. Evidence is presented showing that NO and GAPDH S-nitrosation also mediate cell death during chronological life span pointing to a physiological role of NO in yeast apoptosis.

Glycolytic Enzyme Interactions with Yeast and Skeletal Muscle F-Actin

Interaction of glycolytic enzymes with F-actin is suggested to be a mechanism for compartmentation of the glycolytic pathway. Earlier work demonstrates that muscle F-actin strongly binds glycolytic enzymes, allowing for the general conclusion that “actin binds enzymes”, which may be a generalized phenomenon. By taking actin from a lower form, such as yeast, which is more deviant from muscle actin than other higher animal forms, the generality of glycolytic enzyme interactions with actin and the cytoskeleton can be tested and compared with higher eukaryotes, e.g., rabbit muscle. Cosedimentation of rabbit skeletal muscle and yeast F-actin with muscle fructose-1,6-bisphosphate aldolase (aldolase) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) followed by Scatchard analysis revealed a biphasic binding, indicating high- and low-affinity domains. Muscle aldolase and GAPDH showed low-affinity for binding yeast F-actin, presumably because of fewer acidic residues at the N-terminus of yeast actin; this difference in affinity is also seen in Brownian dynamics computer simulations. Yeast GAPDH and aldolase showed low-affinity binding to yeast actin, which suggests that actin-glycolytic enzyme interactions may also occur in yeast although with lower affinity than in higher eukaryotes. The cosedimentation results were supported by viscometry results that revealed significant cross-linking at lower concentrations of rabbit muscle enzymes than yeast enzymes. Brownian dynamics simulations of yeast and muscle aldolase and GAPDH with yeast and muscle actin compared the relative association free energy. Yeast aldolase did not specifically bind to either yeast or muscle actin. Yeast GAPDH did bind to yeast actin although with a much lower affinity than when binding muscle actin. The binding of yeast enzymes to yeast actin was much less site specific and showed much lower affinities than in the case with muscle enzymes and muscle actin.

Crystallization and preliminary crystallographic analysis of glyceraldehyde 3-phosphate dehydrogenase from Sacchromyces cerevisiae (baker's yeast).

Two related and not thoroughly resolved issues in biochemistry concern the role, if any, of enzyme surfaces in routine metabolism and the method by which metabolic intermediates move between enzyme active sites during multi-step degradation or synthesis. An important enzyme for which a detailed three-dimensional structural analysis has been initiated is yeast glyceraldehyde 3-phosphate dehydrogenase (yGAP-DH). This enzyme is active as a tetramer of total molecular weight of 145 kDa and requires nicotinamide adenine dinucleotide (NAD+) as cofactor. In this report, the crystallization and preliminary crystallographic characterization of several crystal forms of yGAP-DH are described. Of the five distinct crystal forms, the most suitable was found to contain the holo-enzyme, and the crystals were grown by the vapor-diffusion method using polyethylene glycol 6000 as precipitant, sodium acetate as buffer (pH 4.6), and NAD+ and dithiothreitol as additives. The crystals belong to the orthorhombic space group P21212, with cell dimensions of a = 87.33, b = 96.11 and c = 115.34 A. These crystals are mechanically strong, relatively stable in the X-ray beam and diffract X-rays (from a normal rotating-anode radiation source) to better than 2 A resolution. A full 2.1 A resolution diffraction data set (98% completion) has been measured. The three-dimensional structures of related GAP-DH enzymes from several other sources have been determined and reported, and are available for a molecular replacement structure solution.

Expression of mouse cathepsin L cDNA in Saccharomyces cerevisiae: Evidence that cathepsin L is sorted for targeting to yeast vacuole

To investigate the intracellular transport mechanism of lysosomal cathepsin L in yeast cells, we attempted to produce mouse cathepsin L in Saccharomyces cerevisiae by placing the coding region under the control of the promoter of the yeast glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. Immunoblotting analysis by the use of an antibody specific for rat cathepsin L revealed that the yeast cells carrying the cathepsin L coding sequence produced 39- and 30-kDa products, which correspond to the rat procathepsin L and the single-chain form of mature cathepsin L, respectively. The precursor polypeptide showed sensitivity toward endoglycosidase H treatment. Cell fractionation experiments demonstrated that the processed form of 30-kDa cathepsin L was found to be colocalized to the yeast vacuole with the marker enzyme carboxypeptidase Y in a Ficoll step gradient. In the prepared vacuolar fraction, a considerable amount of cathepsin L was revealed to be cofractionated with the vacuolar membranes. Furthermore, the phase separation experiments with Triton X-114 provide the first evidence showing that the mature form of cathepsin L polypeptide is strongly associated with the vacuolar membranes. Therefore, the present results suggest that the mouse cathepsin L precursor polypeptide is initially synthesized as the proenzyme in the yeast cells and then correctly delivered to the vacuole. During the intracellular sorting pathway, the procathepsin L would undergo the post-translational proteolytic processing step to generate the mature enzyme. Based on these lines of evidence, we propose that cathepsin L is recognized by mechanisms similar to those for the intracellular sorting and processing of vacuolar proteins in the yeast cells.

Expression and secretion of goat α-lactalbumin as an active protein in Saccharomyces cerevisiae

An expression plasmid for goat alpha-lactalbumin in Saccharomyces cerevisiae, pSKA100, was constructed into a shuttle vector, pYG100, by inserting cDNA which encodes goat pre-alpha-lactalbumin and two-thirds of the 3'-non-coding region. The goat alpha-lactalbumin was expressed under the yeast glyceraldehyde 3-phosphate dehydrogenase (GPD) promoter and terminator of pYG100 and secreted in the growth medium for yeast as a precise mature protein, possessing specific activity essentially the same as that of authentic goat alpha-lactalbumin in lactose synthesis.

Production of angiotensin-converting enzyme inhibitors from baker's yeast glyceraldehyde-3-phosphate dehydrogenase.

Angiotensin-converting enzyme (ACE) inhibitors were excised from the molecule of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) preparation of baker's yeast by heating at 120 degrees C in 1 M AcOH-20 mM HCl. Three inhibitors were then purified by gel-permeation and reverse-phase chromatographies. One of the yeast ACE inhibitors, YG-3, was GAPDH peptide 79-89 (Pro-Ala-Asn-Leu-Pro-Trp-Gly-Ser-Ser-Asn-Val, IC50:18 microM), and contained the sequence homologous to vertebrate ACE inhibitors (GAPDH peptides 79-86 or 81-88). Other inhibitors, YG-1 (Gly-His-Lys-Ile-Ala-Thr-Phe-Gln-Glu-Arg, IC50: 0.4 microM) and YG-2 (Gly-Lys-Lys-Ile-Ala-Thr-Tyr-Gln-Glu-Arg, IC50: 2 microM), corresponded to amino acid residues 68-77 in two different forms of yeast GAPDH, respectively. Their sequences were quite different from those of the venom peptide family. YG-1 was the most potent ACE inhibitor among yeast and vertebrate GAPDH peptides excised by acid-limited proteolysis. Thus, yeast GAPDH seems to be an excellent source of naturally occurring ACE inhibitors.

Expression and secretion of bovine .BETA.-lactoglobulin in Saccharomyces cerevisiae.

A bovine beta-lactoglobulin (beta-LG) was expressed in Saccharomyces cerevisiae carrying bovine pre-beta-LG cDNA and secreted into its growth medium. The expression plasmid was constructed by inserting the whole coding region of the cDNA encoding pre-beta-LG between the promoter and terminator of the yeast glyceraldehyde 3-phosphate dehydrogenase gene of pYG100, a yeast expression vector. In the supernatant of the yeast growth medium, beta-LG with a native conformation was detected by sandwich ELISA, and its amount was estimated to be 1.1 mg/l. A Western-immunoblotting analysis revealed that beta-LG secrected in the growth medium had the same mobility as that of authentic bovine beta-LG. The N-terminal sequence was also identical with that of authentic mature bovine beta-LG.

Transcriptional mapping of two yeast genes coding for glyceraldehyde 3-phosphate dehydrogenase isolated by sequence homology with the chicken gene

Homology between the coding regions of the chicken and yeast glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes was directly demonstrated by the hybridization of cDNA clone coding for GAPDH in the chicken with EcoRI-digested yeast DNA. A yeast EcoRI fragment library in bacteriophage λ was screened using the chichen cDNA plasmid as probe, and two recombinant phages were isolated, each one containing a different GAPDH gene. The initiation and termination sites for the GAPDH mRNA were localized for the two different GAPDH genes and compared to those of other yeast genes. Measurements of the relative mRNA levels for the two genes show both genes are transcribed at about the same level when yeast are grown on glucose media.

Auramine O as a conformational probe to study glyceraldehyde 3-phosphate dehydrogenase.

The interaction of yeast glyceraldehyde 3-phosphate dehydrogenase with a cationic fluorescent dye, auramine O, was studied by equilibrium dialysis, circular dichroism and fluorescence spectroscopy. Two equal and independent dye binding sites per tetrameric enzyme were detected with a dissociation constant of (4.5 ± 1) × 10−5 M. The maximum fluorescence of bound auramine O is at 515 nm, the excitation spectrum is characterized by the maxima at 380 nm and in the region of 460–470 nm. The complex of auramine O with the enzyme exhibits circular dichroism with a maximum at about 455 nm. The binding of NADH has no effect on the enzyme-dye interaction, whereas the oxidized coenzyme and nicotinamide mononucleotide displace auramine O from the protein. Nicotinamide mononucleotide was shown to induce conformational changes upon binding to the enzyme and to be capable of serving as a coenzyme in the dehydrogenase reaction. AMP, ADP and ADP-ribose form ternary complexes with the enzyme-dye binary complex. This results in a marked increase in fluorescence of the bound dye and was used to characterize the interaction between the enzyme and the adenine-containing NAD+ fragments. In the presence of glyceraldehyde 3-phosphate, the fluorescence of the bound auramine O is quenched, the dye-protein association constant remaining unchanged. Glyceraldehyde, a non-phosphorylated substrate analog, and glycerol 3-phosphate, a competitive inhibitor, are without any effect. Modification of the active-site cysteine-149 with iodoacetate does not affect the dye binding or fluorescence, whereas modification with iodoacetamide diminishes the fluorescence of the enzyme-bound dye without altering the dissociation constant. The effect of adenine-containing NAD+ fragments on the fluorescence of the enzyme-bound dye does not change upon modification of cysteine-149. Auramine O, added together with ADP-ribose, protects the apoenzyme against the inactivation due to modification of essential arginine residues. The effect is similar to that of NAD+ and is absent with auramine O alone or ADP-ribose alone.

Half-of-the-sites reactivity in the catalytic mechanism of yeast glyceraldehyde 3-phosphate dehydrogenase

A half-of-the-sites effect is observed in yeast glyceraldehyde 3-phosphate dehydrogenase for the substrate phosphoglyceroyl phosphate, i.e. only two of the four initially identical subunits are acylated. Excess substrate accelerates the hydrolysis of the phosphoglyceroyl enzyme both in the presence and absence of NAD. This acceleration by excess acyl phosphate is not seen with acetyl phosphate or furyl acryloyl phosphate, even though a half-of-the-sites effect is observed with both. Both the triacetyl and trifuryl acryloyl enzymes can be prepared from the nitrophenyl esters and the hydrolysis of the first acyl group is accelerated in these derivatives. Apparently in all three cases, ligand-induced changes cause the rate of acylation of diacyl enzyme to be decreased and the rate of deacylation of triacyl enzyme to be increased. The balance of the two rates with the true substrate leads to accelerated hydrolysis which is absent for the pseudo-substrates. Thus, ligand-induced co-operativity between subunits has an important effect on the catalytic mechanism.

Reactive lysines of yeast glyceraldehyde 3-phosphate dehydrogenase. Attachment of a reporter group to a specific non-essential residue

The lysine-183 residues of yeast glyceraldehyde 3-phosphate dehydrogenase, in contrast to the cysteine-149 residues, react independently with acylating and alkylating agents. Modification of all four residues is required to inactivate the enzyme in spite of the fact that this residue is apparently in the neighborhood of the cysteine-149 involved in half-of-the-sites activity. The modification of the lysine-183 residue, however, influences the half-of-the-sites effect since alkylation of the cysteine-149 residues of the enzyme whose lysine-183 residues are acetylated follows a linear pattern with each subunit acting independently. Four lysine residues outside the active site can be modified with fluorodinitrobenzene, causing 80% loss in enzyme activity. Once again each subunit acts independently. This same residue can also be modified by a fluorescein label which can serve as a reporter group for binding and conformational changes occurring at the active site. The results add support for the functional symmetry of the apo-enzyme and demonstrate how the co-operativity between subunits can be altered by amino acid modification.

Half-of-the-sites reactivity and negative co-operativity: The case of yeast glyceraldehyde 3-phosphate dehydrogenase

Covalent modification of two of the four cysteine-149 residues of yeast glyceraldehyde 3-phosphate dehydrogenase, at pH 8.5, decreases the reactivity of the remaining two cysteine-149 residues and essentially inactivates the protein. The structure of the modifying reagent has only a secondary influence on this half-of-the-sites effect. Reactivity studies, together with the existing X-ray and sequence studies, suggest that the four active sites are initially functionally identical both in activity and in cysteine reactivity. The half-of-the-sites effect therefore arises in part or in whole from ligand-induced negatively co-operative conformational changes. A detailed kinetic study with iodoacetamide gives relative values of two rapidly reacting groups, a third more slowly reacting, and a fourth very slowly reacting group. These data, added to the existing data on cytidine triphosphate synthetase and alkaline phosphatase, suggest that the half-of-the-sites phenomena in many enzymes may be explained by ligand-induced negative co-operativity triggered by binding or covalent bond formation or both.

Half-of-the-sites reactivity of yeast glyceraldehyde 3-phosphate dehydrogenase

Yeast glyceraldehyde 3-phosphate dehydrogenase is shown to exhibit a half-of-the-sites reactivity with both alkylating and acylating reagents. The shape of the inactivation profiles indicates that disubstituted enzyme is inactive and that monosubstituted enzyme has approximately 10% of the activity of native enzyme.

A Rapid Purification Procedure for Glyceraldehyde 3-Phosphate Dehydrogenase from Bakers' Yeast

Abstract A new procedure utilizing column chromatography has been devised for the purification of yeast glyceraldehyde 3-phosphate dehydrogenase. It yields enzyme of the highest specific activity in a shorter period of time and with a better yield than the traditional procedure. The isozyme isolated is the most abundant and most acidic of the set of isozymes previously identified.

Positive and negative cooperativity in yeast glyceraldehyde 3-phosphate dehydrogenase.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTPositive and negative cooperativity in yeast glyceraldehyde 3-phosphate dehydrogenaseRobert A. Cook and Daniel E. Koshland, Jr.Cite this: Biochemistry 1970, 9, 17, 3337–3342Publication Date (Print):August 1, 1970Publication History Published online1 May 2002Published inissue 1 August 1970https://doi.org/10.1021/bi00819a007RIGHTS & PERMISSIONSArticle Views251Altmetric-Citations116LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (571 KB) Get e-Alerts Get e-Alerts

Reversible dissociation of yeast glyceraldehyde 3-phosphate dehydrogenase by adenosine triphosphate.

Reversible dissociation of yeast glyceraldehyde 3-phosphate dehydrogenase by adenosine triphosphate.

Metabolic control and structure of glycolytic enzymes. VII. Destabilization and inactivation of yeast glyceraldehyde 3-phosphate dehydrogenase by adenosine phosphates and chymotrypsin

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMetabolic control and structure of glycolytic enzymes. VII. Destabilization and inactivation of yeast glyceraldehyde 3-phosphate dehydrogenase by adenosine phosphates and chymotrypsinShih Tzy Yang and W. C. Deal, Jr.Cite this: Biochemistry 1969, 8, 7, 2814–2820Publication Date (Print):July 1, 1969Publication History Published online1 May 2002Published inissue 1 July 1969https://doi.org/10.1021/bi00835a018RIGHTS & PERMISSIONSArticle Views34Altmetric-Citations34LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (783 KB) Get e-Alerts Get e-Alerts