Algal glyceraldehyde-3-phosphate dehydrogenase. Pyridine-nucleotide requirements of two enzymes purified from Scenedesmus obliquus.

Two enzymes with glyceraldehyde-3-phosphate dehydrogenase activity have been purified from heterotrophically grown Scenedesmus obliquus by ion-exchange chromatography and gel filtration. The D-enzyme has a molecular weight of 550000 and a VNADH: VNADPH ratio of 16 whereas the T-enzyme has a molecular weight of 140000 and a VNADH:VNADPH ratio of 0.15. The two enzymes, however, are very similar with regard to their Michaelis constants for the reduced pyridine nucleotides, pH optimum, subunit size and ultraviolet absorption.     

Probing the coenzyme specificity of glyceraldehyde-3-phosphate dehydrogenases by site-directed mutagenesis

By combining our knowledge of the crystal structure of the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the sequence of the photosynthetic NADP-dependent GAPDH of the chloroplast, two particular amino acid residues were predicted as the principal determinants of differing coenzyme specificity. By use of site-directed mutagenesis, the amino acids Leu 187 and Pro 188 of GAPDH from Bacillus stearothermophilus have been replaced with Ala 187 and Ser 188, which occur in the sequence from the chloroplast enzyme. The resulting mutant was shown to be catalytically active not only with its natural coenzyme NAD but also with NADP, thus confirming the initial hypothesis. This approach has not only enabled us to alter the coenzyme specificity by minimal amino acid changes but also revealed factors that control the relative affinity of the enzyme for NAD and NADP.     

Glyceraldehyde-3-phosphate dehydrogenase of Scenedesmus obliquus. Effects of dithiothreitol and nucleotide on coenzyme specificity.

NADH-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.--) of the photosynthetic alga Scenedesmus obliquus is converted to an NADPH specific form by incubation with dithiothreitol. The change in nucleotide specificity is accompanied by a reduction in the molecular weight of the enzyme from 550 000 to 140 000. Prolonged incubation with dithiothreitol results in the further dissociation of the enzyme to an inactive 70 000 dalton species. The 140 000 dalton, NADPH-specific enzyme is stabilized against dissociation and inactivation by the presence of NAD(H) or NADP(H). Optimum stimulation of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase activity is achieved on incubation of the NADH-specific enzyme with dithiothreitol and NADPH, or dithiothreitol and a 1,3-diphosphoglycerate generating system. The relevance of these observations to in vivo light-induced changes in the nucleotide specificity of the enzyme is discussed.     

Quaternary structure of higher plant glyceraldehyde-3-phosphate dehydrogenases.

1. NAD(P)+-induced changes in the aggregational state of prepurified NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) were used to isolate the enzyme from Spinacia oleracea, Pisum sativaum and Hordeum vulgare. Each of the three plant species contains two separate isoenzymes. Isoenzyme 1 (fast moving during conventional electrophoresis) precipitates with the ammonium sulfate fraction 55--70% saturation. It shows two separate subunits in dodecylsulfate gels, which are probably arranged as A2B2 in the native enzyme molecule. Isoenzyme 2 (slow moving during conventional electrophoresis) precipitates with the ammonium sulfate fraction 70--95%. It contains a sigle subunit of the same Mr as subunit A in isoenzyme 1 and is apparently a tetramer (A4). The molecular weights of subunits A/B for spinach, peas and barley were determined as 38,000/40,000, 38,000/42,000 and 36,000/39,000 respectively. 2. The NAD-specific glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) was purified from Spinacia oleracea and Pisum sativum by affinity chromatography on blue Sepharose CL-6B. The enzyme from both plant species is shown to be a tetramer of subunits with Mr 39,000. 3. The present findings contrast with heterogeneous results obtained previously by other authors. These results suggested that there are considerable interspecific differences in the quaternary structure of glyceraldehyde-3-phosphate dehydrogenases from higher plants.     

Engineering the allosteric properties of archaeal non-phosphorylating glyceraldehyde-3-phosphate dehydrogenases

The archaeal non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN, EC 1.2.1.9) is a highly allosteric enzyme activated by glucose 1-phosphate (Glc1P). Recent kinetic analyses of two GAPN homologs from Sulfolobales show different allosteric behaviors toward the substrate glyceraldehyde-3-phosphate (GAP) and the allosteric effector Glc1P. In GAPN from Sulfolobus tokodaii (Sto-GAPN), Glc1P-induced activation follows an increase in affinity for GAP rather than an increase in maximum velocity, whereas in GAPN from Sulfolobus solfataricus (Sso-GAPN), Glc1P-induced activation follows an increase in maximum velocity rather than in affinity for GAP. To explore the molecular basis of this difference between Sto-GAPN and Sso-GAPN, we generated 14 mutants and 2 chimeras. The analyses of chimeric GAPNs generated from regions of Sto-GAPN and Sso-GAPN indicated that a 57-residue module located in the subunit interface was clearly involved in their allosteric behavior. Among the point mutations in this modular region, the Y139R variant of Sto-GAPN no longer displayed a sigmoidal K-type-like allostery, but instead had apparent V-type allostery similar to that of Sso-GAPN, suggesting that the residue located in the center of the homotetramer critically contributes to the allosteric behavior.     

Structural basis for the thermal stability of glyceraldehyde-3-phosphate dehydrogenases

The amino acid sequences of two thermophilic and five mesophilic glyceraldehyde-3-phosphate dehydrogenases have been compared with the known three-dimensional structure of this enzyme to determine the factors responsible for thermal stability. The changes are greatest in the S-loop regions at the center of the tetramer, which show a quantitative increase in hydrophobicity and polarity that can strengthen subunit interactions in a complementary manner. The S-loops also show increases in residue volume and bulk that may indicate a tighter packing at the molecular center. In addition, there are changes in the secondary structural parameters indicating that the helices, in particular, may be more stable in the thermophilic proteins. Increases in the hydrophobicity of domain and subunit contacts for the Thermus aquaticus glyceraldehyde-3-phosphate dehydrogenase may explain why it is the most thermostable protein in this series.     

Prokaryotic features of a nucleus-encoded enzyme. cDNA sequences for chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases from mustard (Sinapis alba)

Two cDNA clones, encoding cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from mustard (Sinapis alba), have been identified and sequenced. Comparison of the deduced amino acid sequences with one another and with the GAPDH sequences from animals, yeast and bacteria demonstrates that nucleus-encoded subunit A of chloroplast GAPDH is distinct from its cytosolic counterpart and the other eukaryotic sequences and relatively similar to the GAPDHs of thermophilic bacteria. These results are compatible with the hypothesis that the nuclear gene for subunit A of chloroplast GAPDH is of prokaryotic origin. They are in puzzling contrast with a previous publication demonstrating that Escherichia coli GAPDH is relatively similar to the eukaryotic enzymes [Eur. J. Biochem. 150, 61-66 (1985)].     

Comparative analysis of two glyceraldehyde-3-phosphate dehydrogenases from a thermoacidophilic archaeon,Sulfolobus tokodaii

Sulfolobus tokodaii, a thermoacidophilic archaeon, possesses two structurally and functionally different enzymes that catalyze the oxidation of glyceraldehyde-3-phosphate (GAP): non-phosphorylating GAP dehydrogenase (St-GAPN) and phosphorylating GAP dehydrogenase (St-GAPDH). In contrast to previously characterized GAPN from Sulfolobus solfataricus, which exhibits V-type allosterism, St-GAPN showed K-type allosterism in which the positive cooperativity was abolished with concomitant activation by glucose 1-phosphate (G1P). St-GAPDH catalyzed the reversible oxidation of GAP to 1,3-bisphosphoglycerate (1,3-BPG) with high gluconeogenic activity, which was specific for NADPH, while both NAD⁺ and NADP⁺ were utilized in the glycolytic direction.Structured summary of protein interactionsGAPDH and GAPDH bind by molecular sieving (View interaction) GAPN and GAPN bind by 2.2molecular sieving (View interaction).     

Sequence comparison of glyceraldehyde-3-phosphate dehydrogenases from the three urkingdoms: evolutionary implication

The primary structure of the glyceraldehyde-3-phosphate dehydrogenase from the archaebacteria shows striking deviation from the known sequences of eubacterial and eukaryotic sequences, despite unequivocal homologies in functionally important regions. Thus, the structural similarity between the eubacterial and eukaryotic enzymes is significantly higher than that between the archaebacterial enzymes and the eubacterial and eukaryotic enzymes. This preferred similarity of eubacterial and eukaryotic glyceraldehyde-3-phosphate dehydrogenase structures does not correspond to the phylogenetic distances among the three urkingdoms as deduced from comparisons of ribosomal ribonucleic acid sequences. Indications will be presented that the closer relationship of the eubacterial and eukaryotic glyceraldehyde-3-phosphate dehydrogenase resulted from a gene transfer from eubacteria to eukaryotes after the segregation of the three urkingdoms.     

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase of Candida albicans is a surface antigen.

A lambda gt11 cDNA library from Candida albicans ATCC 26555 was screened by using pooled sera from two patients with systemic candidiasis and five neutropenic patients with high levels of anti-C. albicans immunoglobulin M antibodies. Seven clones were isolated from 60,000 recombinant phages. The most reactive one contained a 0.9-kb cDNA encoding a polypeptide immunoreactive only with sera from patients with systemic candidiasis. The whole gene was isolated from a genomic library by using the cDNA as a probe. The nucleotide sequence of the coding region showed homology (78 to 79%) to the Saccharomyces cerevisiae TDH1 to TDH3 genes coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and their amino acid sequences showed 76% identity; thus, this gene has been named C. albicans TDH1. A rabbit polyclonal antiserum against the purified cytosolic C. albicans GAPDH (polyclonal antibody [PAb] anti-CA-GAPDH) was used to identify the GAPDH in the beta-mercaptoethanol extracts containing cell wall moieties. Indirect immunofluorescence demonstrated the presence of GAPDH at the C. albicans cell surface, particularly on the blastoconidia. Semiquantitative flow cytometry analysis showed the sensitivity of this GAPDH form to trypsin and its resistance to be removed with 2 M NaCl or 2% sodium dodecyl sulfate. The decrease in fluorescence in the presence of soluble GAPDH indicates the specificity of the labelling. In addition, a dose-dependent GAPDH enzymatic activity was detected in intact blastoconidia and germ tube cells. This activity was reduced by pretreatment of the cells with trypsin, formaldehyde, and PAb anti-CA-GAPDH. These observations indicate that an immunogenic, enzymatically active cell wall-associated form of the glycolytic enzyme GAPDH is found at the cell surface of C. albicans cells.     

Circular permutation within the coenzyme binding domain of the tetrameric glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.

A circularly permuted (cp) variant of the phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus has been constructed with N- and C-termini created within the coenzyme binding domain. The cp variant has a kcat value equal to 40% of the wild-type value, whereas Km and KD values for NAD show a threefold decrease compared to wild type. These results indicate that the folding process and the conformational changes that accompany NAD binding during the catalytic event occur efficiently in the permuted variant and that NAD binding is tighter. Reversible denaturation experiments show that the stability of the variant is only reduced by 0.7 kcal/mol compared to the wild-type enzyme. These experiments confirm and extend results obtained recently on other permuted proteins. For multimeric proteins, such as GAPDH, which harbor subunits with two structural domains, the natural location of the N- and C-termini is not a prerequisite for optimal folding and biological activity.     

Characterization of glyceraldehyde-3-phosphate dehydrogenase as a novel transferrin receptor

A majority of cells obtain of transferrin (Tf) bound iron via transferrin receptor 1 (TfR1) or by transferrin receptor 2 (TfR2) in hepatocytes. Our study establishes that cells are capable of acquiring transferrin iron by an alternate pathway via GAPDH.These findings demonstrate that upon iron depletion, GAPDH functions as a preferred receptor for transferrin rather than TfR1 in some but not all cell types. We utilized CHO-TRVb cells that do not express TfR1 or TfR2 as a model system. A knockdown of GAPDH in these cells resulted in a decrease of not only transferrin binding but also associated iron uptake. The current study also demonstrates that, unlike TfR1 and TfR2 which are localized to a specific membrane fraction, GAPDH is located in both the detergent soluble and lipid raft fractions of the cell membrane. Further, transferrin uptake by GAPDH occurs by more than one mechanism namely clathrin mediated endocytosis, lipid raft endocytosis and macropinocytosis. By determining the kinetics of this pathway it appears that GAPDH-Tf uptake is a low affinity, high capacity, recycling pathway wherein transferrin is catabolised. Our findings provide an explanation for the detailed role of GAPDH mediated transferrin uptake as an alternate route by which cells acquire iron.     

Acceleration of glycolysis in the presence of the non-phosphorylating and the oxidized phosphorylating glyceraldehyde-3-phosphate dehydrogenases

Mild oxidation of glyceraldehyde-3-phosphate dehydrogenase in the presence of hydrogen peroxide leads to oxidation of some of the active site cysteine residues to sulfenic acid derivatives, resulting in the induction of acylphosphatase activity. The reduced active sites of the enzyme retain the ability to oxidize glyceraldehyde-3-phosphate yielding 1,3-diphosphoglycerate, while the oxidized active sites catalyze irreversible cleavage of 1,3-diphosphoglycerate. It was assumed that the oxidation of glyceraldehyde-3-phosphate dehydrogenase by different physiological oxidants must accelerate glycolysis due to uncoupling of the reactions of oxidation and phosphorylation. It was shown that the addition of hydrogen peroxide to the mixture of glycolytic enzymes or to the muscle extract increased production of lactate, decreasing the yield of ATP. A similar effect was observed in the presence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase catalyzing irreversible oxidation of glyceraldehyde-3-phosphate into 3-phosphoglycerate. A role of glyceraldehyde-3-phosphate dehydrogenase in regulation of glycolysis is discussed.     

Influence of coenzyme on the refolding and reassociation in vitro of glyceraldehyde-3-phosphate dehydrogenase from yeast.

Kinetic analysis of the reactivation in vitro of glyceraldehyde-3-phosphate dehydrogenase from yeast in the presence of NAD+ suggested that transconformation reactions of inactive monomers and their subsequent association to native tetramers are responsible for the sigmoidal relaxations [R. Rudolph et al. (1977) Eur. J. Biochem. 81, 563-570]. Comparison with the reactivation behaviour in the absence of coenzyme was not feasible at this stage due to the instability of the apoenzyme. In the present study, solvent conditions were established which allowed both apoenzyme and holoenzyme to exhibit high stability. The apoenzyme is stable in phosphate buffer; but if excess NAD+ and phosphate are present (both of which stabilize the enzyme if applied separately), destabilization occurs. Protection of functional groups against oxidation by addition of a reducing agent and by degassing and preventing contact with air, increase the stability. Only partial stabilization can be achieved in the presence of NADH. Comparing the kinetics of reactivation in the presence and absence of coenzymes shows that both oxidized and reduced coenzyme enhance the rate of reactivation significantly, and to the same extent. The kinetic effect of coenzyme binding to the refolding polypeptide chain is discussed in terms of the stabilization of intermediates or end products of reconstitution on the one hand, and acceleration of folding and association reactions, on the other.     

The NADP-linked glyceraldehyde-3-phosphate dehydrogenases of Anabaena variabilis and Synechocystis PCC 6803, which lack one of the cysteines found in the higher plant enzyme,are not reductively activated

Light activation of NADP-linked glyceraldehyde-3-P dehydrogenase involves reductive cleavage of a disulfide bond. We have proposed that the inactivating disulfide locks the two domains of the enzyme, preventing catalysis, and we have tentatively identified the two critical cysteine residues in the chloroplast enzyme (D. Li, F.J. Stevens, M. Schiffer and L.E. Anderson (1994) Biophys J. 67: 29–35). We reasoned that if activation of this enzyme involves these cysteines that enzymes lacking one or both should be active in the dark and insensitive to reductants. One of these cysteines is present in the enzymes from Anabaena variabilis and Synechocystis PCC 6803 but the other is not. Consistent with the proposed mechanism, glyceraldehyde-3-P dehydrogenase is not affected by DTT-treatment in extracts of either of these cyanobacteria. Fructosebisphosphatase is DTT-activated in extracts of both of these cyanobacteria and glucose-6-P dehydrogenase is inactivated in Synechocystis, as in higher plant chloroplasts. Apparently reductive modulation is possible in these cyanobacteria but glyceraldehyde-3-P dehydrogenase is not light activated.