Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.2.1.13 (glyceraldehyde-3-phosphate dehydrogenase)
 6,511 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The structural and functional properties of chloroplast glyceraldehyde-3-P-dehydrogenase I (D-Glyceraldehyde-3-phosphate: NADP oxidoreductase (phosphorylating) EC 1.2.1.13) from Spinacia oleracea were investigated by limited proteolysis. The enzyme is insensitive to trypsin and chymotrypsin, while Staphylococcus aureus V8 protease cleaves the C-terminal region of its subunits. Subunit A (36 kDa) is only partially cleaved at Glu 317. No intact subunit B (39 kDa) is found at the end of the proteolytic experiment: two forms are originated from this subunit which is cleaved at Glu 342 and Glu 320. Proteolytic cleavage at these sites does not significantly alter enzymatic activity, but leads to destabilization of the protein. Unlike the intact parent enzyme (600 kDa) the cleaved enzyme behaves as a 150-kDa species in size exclusion chromatography.
 ...
PMID:Limited proteolysis of chloroplast glyceraldehyde-3-phosphate dehydrogenase (NADP) from Spinacia oleracea. 835 35

On the basis of the three-dimensional structure of the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and of sequence comparison with the photosynthetic NAD(P)-dependent GAPDH of the chloroplast, a series of mutants of GAPDH from Bacillus stearothermophilus have been constructed. The results deduced from kinetic and binding studies suggest that the absence of activity of the wild-type GAPDH with NADP as a cofactor is the consequence of at least three factors: (1) steric hindrance, (2) electrostatic repulsion between the charged carboxyl group of Asp32 and the 2'PO4, and (3) structural determinants at the subunit interface of the tetramer. The best value for kcat/KM and KD for NADP was observed for the D32A-L187A-P188S mutant. This triple mutation leads to a switch in favor of NADP specificity but with a kcat/KM ratio 50- and 80-fold less than that observed for the wild type with NAD and for the chloroplast GAPDH with NADP, respectively. Substituting the invariant chloroplastic Thr33-Gly34-Gly35 for the B. stearothermophilus Leu33-Thr34-Asp35 residues on the double mutant Ala187-Ser188 does not improve significantly the affinity for NADP while substituting Ala32 for Asp32 on the double mutant does. Clearly, other subtle adjustments in the adenosine subsite are needed to reconcile the presence of the carboxylate group of Asp32 and the 2'-phosphate of NADP. Kinetic studies indicate a change of the rate-limiting step for the mutants. This could be the consequence of an incomplete apo-holo transition.(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:Determinants of coenzyme specificity in glyceraldehyde-3-phosphate dehydrogenase: role of the acidic residue in the fingerprint region of the nucleotide binding fold. 839 44

The overlapping genes encoding phosphoglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehydrogenase (GraP-DH) from the hyperthermophilic archaeon Sulfolobus solfataricus have been cloned and sequenced. PCR primers based on highly conserved regions of different PGK sequences were used to isolate an internal region of the pgk gene. This was then used to screen a genomic library to isolate the full length pgk gene. A 2.5-kb BglII fragment of S. solfataricus DNA contained both the pgk gene and the gap gene immediately downstream. Unexpectedly, the pgk and gap genes were found to overlap by 8 bp, with the initiation codon of the gap gene preceding the termination codon of the pgk gene. Evidence that the two genes are co-transcribed was obtained by Northern-blot analysis. The S. solfataricus PGK amino acid sequence shows 43% and 45% identity to the PGK sequences of the Archaea Methanobacterium bryantii and Methanothermus fervidus, respectively. High level expression of the S. solfataricus PGK and GraP-DH in Escherichia coli was achieved, with heat treatment at 80 degrees C proving an effective first step in the purification of these recombinant enzymes from extracts of the E. coli host. Purified recombinant S. solfataricus PGK and GraP-DH showed half lives of 39 min and 17 h, respectively, at 80 degrees C. Unlike bacterial GraP-DH enzymes, S. solfataricus GraP-DH was able to use both NAD+ and NADP+ as cofactors, but exhibited a marked preference for NADP+.
 ...
PMID:The phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase genes from the thermophilic archaeon Sulfolobus solfataricus overlap by 8-bp. Isolation, sequencing of the genes and expression in Escherichia coli. 852 45

Spinach chloroplast NAD(P)-glyceraldehyde-3-phosphate dehydrogenase (NAD(P)-GAPDH; EC, 1.2.1.13) was purified as the 600-kDa oligomer of low specific activity. Incubation of the enzyme with either a reductant or a 1,3-bisphosphoglycerate (1,3bisPGA) generating system, but most effectively with both, resulted in an increase of the apparent NADPH-dependent activity. Only the 1,3bisPGA treatment caused dissociation and yielded the 150-kDa heterotetramer (A2B2). The higher activity of the tetramer is largely due to a decreased KM value for the substrate 1,3bisPGA. Reductive treatment alone does not dissociate the enzyme. Reduction was equally effective with glutathione as with dithiothreitol or with reduced thioredoxin f. The concentration of 1,3bisPGA required to obtain 50% activity (K alpha) was 19.5 +/- 4.1 microM for the untreated enzyme and 2.0 +/- 1.4 microM for the thiol-pretreated enzyme. Thus, in vitro 1,3bisPGA, alone or--at much lower concentrations--together with a reductant can activate (and dissociate) NAD(P)-GAPDH. The enzyme exhibits similar K alpha values in its reduced and its oxidized form for ATP (1-2 mM), NADP (50-200 microM), and NADPH (0.3-0.5 mM) as positive effectors, but these effectors do not lead to any activation when present together with 0.14 mM NAD. Only 1,3bisPGA retained its characteristic effect in the presence of NAD. The dissociated enzyme reaggregates upon removal of the positive effectors. From these results it is concluded (i) that the role of the reduction of the NAD(P)-GAPDH in vivo is to increase its sensitivity toward the activator 1,3bisPGA and (ii) that the actual activation (and aggregation) state of the enzyme in chloroplasts in the light is regulated by the concentration of 1,3bisPGA as activator in the stroma and its actual activity by the availability of 1,3bisPGA as substrate.
 ...
PMID:Reductive modification and nonreductive activation of purified spinach chloroplast NADP-dependent glyceraldehyde-3-phosphate dehydrogenase. 855 10

Binding of NAD(P)+ to wild type and a series of mutants of the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus designed to alter the cofactor specificity [Clermont, S., Corbier, C., Mely, Y., Gerard, D., Wonacott, A., & Branlant, G. (1993) Biochemistry 21, 10178-10184] has been studied by 31P NMR. In the mutants with the L187A and P188S substitutions, the pyrophosphate signals are split, and the upfield resonance has been assigned to the P(a) phosphate. Titration of the NADP+ 2'-phosphate pKa deduced from its chemical shift shows that the electrostatic environment in the binding site is largely affected by the single point mutations. pKas ranging from 7.7 for the L187A-P188S mutant to < 5.7 for the D32G-L187A-P188S and D32A-L187A-P188S mutants have been observed, thus indicating that the binding of NADP+ is modulated by the ionization state of its 2'-phosphate. In the quintuple mutant L33T-T34G-D35G-L187A-P188S, designed in comparison with the photosynthetic NAD(P)-dependent GAPDH of the chloroplast, the 2'-phosphate has a pKa of 6.8. As further stabilizing interactions like hydrogen bonds or positively charged side chains would lower this pKa, it is suggested that the 2'-phosphate ionization state of bound NADP+ in chloroplastic GAPDH is dianionic. The NADP+ dissociation rate constants (k(off)) of the three mutants D32G, L187A-P188S, and D32G-L187A-P188S, are higher at pH 6.1 than at pH 8.1 and are similar at the same pH, indicating that the difference in binding affinity between these three mutants results from the molecular recognition step or conformational change upon binding.
 ...
PMID:Phosphorus-31 nuclear magnetic resonance studies on coenzyme binding and specificity in glyceraldehyde-3-phosphate dehydrogenase. 863 48

NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been purified to electrophoretic homogeneity from Synechococcus PCC 7942 cells. The native enzyme had a molecular mass of 160 kDa and consisted of four subunits with a molecular mass of 41 kDa. The activity was 6-fold higher with NADPH than with NADH; the apparent Km values for NADPH and NADH were 62 +/- 4.5 and 420 +/- 10.5 microM respectively. The gene encoding NADP-dependent GAPDH was cloned from the chromosomal DNA of Synechococcus 7942. A 1140 bp open reading frame, encoding an enzyme of 380 amino acid residues (approx.molecular mass of 41.3 kDa) was observed. The deduced amino acid sequence of the gene had a greater sequence similarity to the NADP-dependent and chloroplastic form than to the NAD-dependent and cytosolic form. The Synechococcus 7942 enzyme lacked one of the cysteines involved in the light-dependent regulation of the chloroplast enzymes of higher plants. The recombinant enzyme expressed in Escherichia coli as well as the native enzyme purified from Synechococcus 7942 cells were resistant to 1 mM H2O2.
 ...
PMID:Enzymic and molecular characterization of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Synechococcus PCC 7942: resistance of the enzyme to hydrogen peroxide. 868 18

Chloroplast NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase (NAD(P)-GAPDH; EC 1.2.1.13) consists of two types of subunits: GapA and GapB, which are rather similar, except that GapB carries an unique C-terminal sequence extension. Here, we report evidence that this sequence extension might be responsible for aggregation and dark inactivation of the enzyme in vivo. Recently, it had been demonstrated that upon limited proteolysis of the purified 600 kDa enzyme, using the Staphylococcus aureus V8 endoproteinase (Zapponi et al. (1993) Biol. Chem. Hoppe-Seyler 374, 395-402), the C-terminus of GapB can be removed, giving rise to the 150 kDa form. Based on these findings, we analyzed the changed catalytic properties of the enzyme after proteolysis and its ability to reaggregate. The time-course of proteolysis is paralleled by a strong increase in enzyme activity and the appearance of the tetrameric enzyme form, the increase of apparent activity preceding disaggregation. The proteolyzed enzyme is characterized by its increased affinity towards the substrate 1,3-bisphosphoglycerate and thus resembles the fully activated intact enzyme. In contrast to the effector-mediated activation of the intact enzyme, both proteolytic activation and the resulting disaggregation of the high-molecular-weight form cannot be reversed, even by incubation with NAD.
 ...
PMID:C-terminal truncation of spinach chloroplast NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase prevents inactivation and reaggregation. 881 30

The objective of this study was to determine whether the concentration of pyridine nucleotides in muscle and liver tissue of quail affected the heat stability of aldolase and selected enzymes involved in the oxidation-reduction of these cofactors. The thermal stability of malic enzyme, glyceraldehyde-3-phosphate dehydrogenase, lactic dehydrogenase, and aldolase in liver, and in pectoral muscle of quail was studied at incubation temperatures ranging from 27 to 60 degrees C. The concentrations of liver NAD, NADP, NADPH and the thermal inactivation of liver malic enzyme, glyceraldehyde-3-phosphate dehydrogenase, lactic dehydrogenase, and aldolase were not affected by niacin deficiency. In contrast, pectoral muscle glyceraldehyde-3-phosphate dehydrogenase in the niacin deficient quail compared to that of the controls had a markedly reduced thermal stability. This was associated with a corresponding decrease in the concentration of NAD and possibly NADPH. However, lactic dehydrogenase and aldolase activities were not affected. A similar pattern of heat inactivation was obtained when dialysed muscle and liver extracts were spiked with NAD or NADP. In these studies, NAD(P) protected muscle glyceraldehyde-3-phosphate dehydrogenase against heat inactivation to a much greater degree than that obtained with the other enzymes from muscle or liver tissue. These results suggest a causative relationship between the thermal stability of glyceraldehyde-3-phosphate dehydrogenase and coenzyme status in pectoral muscle tissue. This effect of niacin deficiency on the thermal stability of enzymes appears to be quite selective and specific.
 ...
PMID:Effect of niacin deficiency on the thermal stability of NAD- and NADP-dependent dehydrogenases in liver and pectoral muscle of Japanese quail. 893 Jan 42

Higher-plant chloroplast NAD(P)-glyceraldehyde 3-phosphate dehydrogenase (NAD(P)-GAPDH; EC 1.2.1.13) is composed of two different nuclear-encoded subunits, GAPA and GAPB, forming the highly active heterotetrameric A2B2 enzyme. The main difference between these two subunits is a C-terminal extension of about 30 amino acid residues of GAPB. We present cDNA clones for a nuclear-encoded chloroplast protein from pea, spinach and tobacco, which we have named CP12. The mature protein consists of only 74, 75 and 76 amino acid residues, respectively and contains two domains with significant homology to the C-terminal extension of GAPB. Affinity chromatography approaches reveal also a specific interaction between CP12 and chloroplast GAPDH. Northern blot analysis indicates that CP12 is, like plastid GAPDH, expressed in green and also in etiolated leaves. Further homology is observed between CP12 and ORF3, an open reading frame located in the hox gene cluster of Anabaena variabilis. This gene cluster encodes the subunits of the bidirectional NADP(+)-dependent [NiFeS] dehydrogenase. We propose therefore a common evolutionary origin of CP12 and higher-plant chloroplast GAPDH subunit GAPB from the cyanobacterial ORF3.
 ...
PMID:CP12: a small nuclear-encoded chloroplast protein provides novel insights into higher-plant GAPDH evolution. 898 May 47

The function of the membrane-bound ATPase in S. mutans is to regulate cytoplasmic pH values for the purpose of maintaining delta pH. Previous studies have shown that as part of its acid-adaptive ability, S. mutans is able to increase H(+)-ATPase levels in response to acidification. As part of the study of ATPase regulation in S. mutans, we have cloned the ATPase operon and determined its genetic organization. The structural genes from S. mutans were found to be in the order: c, a, b, delta, alpha, gamma, beta, and epsilon; where c and a were reversed from the more typical bacterial organization. The operon contained no I gene homologue but was preceded by a 239-bp intergenic space. Deduced aa sequences from open reading frames indicated that genes encoding homologues of glycogen phosphorylase and nonphosphorylating, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase flank the H(+)-ATPase operon, 5' and 3' respectively. Sequence analysis indicated the presence of three inverted-repeat nt sequences in the glgP-uncE intergenic space. Primer extension analysis of mRNAs prepared from batch-grown or steady-state cultures demonstrated that the transcriptional start site did not change as a function of culture pH value. The data suggest that potential stem-and-loop structures in the promoter region of the operon do not function to alter the starting position of ATPase-specific mRNA transcription.
 ...
PMID:Cloning and nucleotide sequence analysis of the Streptococcus mutans membrane-bound, proton-translocating ATPase operon. 899 91


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>