Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.2.1.13 (glyceraldehyde-3-phosphate dehydrogenase)
6,511 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A mutant of Bacillus stearothermophilus D-glyceraldehyde-3-phosphate dehydrogenase, Ser148----Ala, was produced by oligonucleotide-directed mutagenesis. The study of the catalytic properties of this mutant has shown that this mutation significantly affects the Michaelis constant of inorganic phosphate and to a lesser extent that of 1,3-diphosphoglycerate and D-glyceraldehyde-3-phosphate. This result is consistent with model-building studies which show that, for the phosphorylation step of catalysis, inorganic phosphate must bind to the anion recognition site designated Pi with the C(3) phosphate of the acyl-enzyme intermediate in the alternative anion site Ps. Studies of the enantiomeric specificity using D- and L-glyceraldehyde as substrates show that the hydroxyl group of Ser148, combined with the presence of the C(3) phosphate of the substrate, enhances stereospecificity as well as catalysis. However, the stereospecific effect cannot be a consequence of the direct interaction of Ser148 with the C(2)-hydroxyl of the substrate. The changed Km for glyceraldehyde-3-phosphate suggests that the initial step of hemithioacetal formation may take place with its C(3) phosphate bound in the Pi site. This supports the molecular mechanism proposed by Moody (1984). Therefore, catalysis could be enhanced through interactions of the serine hydroxyl group not only with inorganic phosphate but also with the C(3) phosphate of glyceraldehyde-3-phosphate.
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PMID:Site-directed mutagenesis of glyceraldehyde-3-phosphate dehydrogenase reveals the role of residue Ser148. 250 80

The structural gene (gapST) encoding glyceraldehyde-3-phosphate dehydrogenase (GPDH; EC 1.2.1.12) from Bacillus stearothermophilus has been cloned in Escherichia coli using plasmid pBR322 as a vector; the homologous gene (gapCO) from Bacillus coagulans was cloned from a phage lambda library. Expression of the cloned gap genes revealed that, as in the wild-type (wt) organisms, the GPDH from B. stearothermophilus (GPDH-ST) was intrinsically heat stable (hs) and that from B. coagulans (GPDH-CO) heat labile (hl). The cloned gap genes were sequenced and the deduced amino acid (aa) sequences were found to be highly conserved (91.6% homology), despite the large difference in thermostability between these two enzymes. Of the 28 aa which differ between the two proteins, most of which occur in the middle third of the monomeric subunit, 5 aa involve replacement of alanine in the hl GPDH-CO, by proline in the hs GPDH-ST, and are especially interesting in terms of their potential contributions to thermostability. Conservation at the DNA level is equally dramatic, with the two gap genes exhibiting 93.3% nucleotide sequence homology. These highly expressed genes exhibit an equivalent codon bias, which more closely resembles that of highly expressed E. coli genes, than that of B. stearothermophilus genes whether highly or weakly expressed.
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PMID:Nucleotide sequences of genes encoding heat-stable and heat-labile glyceraldehyde-3-phosphate dehydrogenases; amino acid sequence and protein thermostability. 222 48

A second thioredoxin, distinct from the one reported by Meng and Hogenkamp in 1981 (J. Biol. Chem. 256, 9174-9182), has been purified to homogeneity from an Escherichia coli strain containing a plasmid encoding a Corynebacterium nephridii thioredoxin. Thioredoxin genes from C. nephridii were cloned into the plasmid pUC13 and transformants were identified by complementation of a thioredoxin negative (trxA-) E. coli strain. The abilities of the transformants to support the growth of several phages suggested that more than one thioredoxin had been expressed [Lim et al. (1987) J. Biol. Chem. 262, 12114-12119]. In this paper we present the purification and characterization of one of these thioredoxins. The new thioredoxin from C. nephridii, designated thioredoxin C-2, is a heat-stable protein containing three cysteine residues/molecule. It serves as a substrate for C. nephridii thioredoxin reductase and E. coli and Lactobacillus leichmannii ribonucleotide reductases. Thioredoxin C-2 catalyzes the reduction of insulin disulfides by dithiothreitol or by NADPH and thioredoxin reductase and is a hydrogen donor for the methionine sulfoxide reductase of E. coli. Spinach malate dehydrogenase (NADP+) and phosphoribulokinase are activated by this thioredoxin while glyceraldehyde-3-phosphate dehydrogenase (NADP+) is not. Like the thioredoxin first isolated from C. nephridii, this new thioredoxin is not a reducing substrate for the C. nephridii ribonucleotide reductase. The complete primary sequence of this second thioredoxin has been determined. The amino acid sequence shows a high degree of similarity with other thioredoxins. Surprisingly, in contrast to the other sequences, this new thioredoxin contains the tetrapeptide -Cys-Ala-Pro-Cys- at the active site. With the exception of the T4 thioredoxin, this is the first example of a thioredoxin that does not have the sequence -Cys-Gly-Pro-Cys-. Our results suggest that, like plant cells, bacterial cells may utilize more than one thioredoxin.
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PMID:Purification, characterization and revised amino acid sequence of a second thioredoxin from Corynebacterium nephridii. 291 72

We have identified the site labeled by arylazido-beta-alanyl-NAD+ (A3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NAD+) in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase by microsequencing and fast atom bombardment mass spectrometry. This NAD+ photoaffinity analogue has been previously demonstrated to modify glyceraldehyde-3-phosphate dehydrogenase in a very specific manner and probably at the active site of the enzyme [Chen, S., Davis, H., Vierra, J. R., & Guillory, R. J. (1984) Biochem. Biophys. Stud. Proteins Nucleic Acids, Proc. Int. Symp., 3rd, 407-425]. The label is associated exclusively with a tryptic peptide that has the sequence Ile-Val-Ser-Asn-Ala-Ser-Cys-Thr-Thr-Asn. In comparison to the amino acid sequence of glyceraldehyde-3-phosphate dehydrogenase from other species, this peptide is in a highly conserved region and is part of the active site of the enzyme. The cysteine residue at position seven was predominantly labeled and suggested to be the site modified by arylazido-beta-alanyl-NAD+. This cysteine residue corresponds to the Cys-149 in the pig muscle enzyme, which has been shown to be an essential residue for the enzyme activity. The present investigation clearly demonstrates that arylazido-beta-alanyl-NAD+ is a useful photoaffinity probe to characterize the active sites of NAD(H)-dependent enzymes.
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PMID:Identification of the arylazido-beta-alanyl-NAD+-modified site in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase by microsequencing and fast atom bombardment mass spectrometry. 377 66

The fasting hypoglycemia (1.78 +/- 0.29 mmol/l) which develops in 48-h-old pigs is partially reversed (3.85 +/- 0.55 mmol/l) after gastric administration of long-chain triglycerides (LCT). The increase in blood glucose induced by LCT feeding was not secondary to a decreased glucose utilization because glucose disappearance rate increased in LCT-fed piglets but resulted from a twofold increase in glucose appearance. By using the crossover-plot technique, the stimulation of hepatic gluconeogenesis induced by LCT feeding has been localized at 1) the level of pyruvate carboxylase owing to the twofold increase in hepatic acetyl-CoA concentration and 2) the level of glyceraldehyde-3-phosphate dehydrogenase secondary to the increase in reducing equivalents (NADH), which displaces this equilibrium reaction in the direction of gluconeogenesis. As blood lactate, pyruvate, and alanine concentrations increased after LCT feeding, the possible effects of LCT on pyruvate dehydrogenase in peripheral tissues are discussed. These data demonstrate that fatty acids stimulate hepatic gluconeogenesis in 48-h-old fasting piglets and underline the role of fat provision in the regulation of glucose homeostasis during the neonatal period in the pig.
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PMID:Effect of intragastric triglyceride administration on glucose homeostasis in newborn pigs. 389 65

Extreme codon bias is seen for the Saccharomyces cerevisiae genes for the fermentative alcohol dehydrogenase isozyme I (ADH-I) and glyceraldehyde-3-phosphate dehydrogenase. Over 98% of the 1004 amino acid residues analyzed by DNA sequencing are coded for by a select 25 of the 61 possible coding triplets. These preferred codons tend to be highly homologous to the anticodons of the major yeast isoacceptor tRNA species. Codons which necessitate site by side GC base pairs between the codons and the tRNA anticodons are always avoided whenever possible. Codons containing 100% G, C, A, U, GC, or AU are also avoided. This provides for approximately equivalent codon-anticodon binding energies for all preferred triplets. All sequenced yeast genes show a distinct preference for these same 25 codons. The degree of preference varies from greater than 90% for glyceraldehyde-3-phosphate dehydrogenase and ADH-I to less than 20% for iso-2 cytochrome c. The degree of bias for these 25 preferred triplets in each gene is correlated with the level of its mRNA in the cytoplasm. Genes which are strongly expressed are more biased than genes with a lower level of expression. A similar phenomenon is observed in the codon preferences of highly expressed genes in Escherichia coli. High levels of gene expression are well correlated with high levels of codon bias toward 22 of the 61 coding triplets. As in yeast, these preferred codons are highly complementary to the major cellular isoacceptor tRNA species. In at least four cases (Ala, Arg, Leu, and Val), these preferred E. coli codons are incompatible with the preferred yeast codons.
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PMID:Codon selection in yeast. 703 77

The cytosolic phosphate potential was estimated in isolated rat liver parenchymal cells incubated with various gluconeogenic substrates. The value of the cytosolic [ATP]/[ADP][Pi] ratio was either estimated directly from measurements of ATP, ADP and Pi after digitonin fractionation of the cells, or calculated by the metabolite indicator method. When cells were incubated with lactate, pyruvate or alanine so that net flux through the indicator enzymes was in gluconeogenic direction, there was excellent agreement between the values obtained by the two methods over a wide range of fluxes. However, when the cells were incubated with substrates that could be converted both to glucose and to lactate so that net flux through the indicator enzymes was in the glycolytic direction, a large difference in the values of the cytosolic [ATP]/([ATP][Pi]) ratio as derived by the two methods was observed. It is concluded that the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase plus 3-phosphoglycerate kinase is out of equilibrium when flux through the reaction is the glycolytic direction, and that use of the metabolite indicator method for the calculation of the cytosolic phosphate potential under these conditions leads to erroneous values.
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PMID:An evaluation of the metabolite indicator method for determining the cytosolic phosphate potential in rat liver cells. 713 15

The unusual thermolability of glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile Bacillus coagulans KU (Crabb et al., Biochemistry 16:4840-4847, 1977) has provided the first opportunity to study a homologous enzyme from the same genus that exhibits a marked difference in thermostability. In pursuit of the structural bases for the thermostability of proteins, the sequences of the amino terminus (residues 1 through 27) and the active-site cysteine cyanogen bromide peptide (residues 130 through 167) of this enzyme have been determined and compared with sequences of the enzyme from other sources. The importance of comparing phylogenetically related proteins is evident from the 87% identity found between these sequences in the enzyme from B. coagulans and Bacillus stearothermophilus, versus only 45% identity for all other known sequences. The marked sequence identity of the enzyme from the two Bacillus species drew attention to the variable region (residues 138 through 140a) which is exposed to the exterior of the quaternary structure of this enzyme. Based on the reported crystallographic structures of the enzyme from lobster muscle and B. stearothermophilus and space-filling models of the variable region, the segment Asp-Pro-Lys-Ala in B. stearothermophilus should be more thermostable than the analogous sequence, Asp-Ala-Ala-Asn, from B. coagulans. In addition, the space-filling models suggested that the spatial relationship of an amino acid side chain and its potential for close packing and interactions with neighboring side chains may be more important than the type of amino acid substituted.
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PMID:Sequence homology in the amino-terminal and active-site regions of thermolabile glyceraldehyde-3-phosphate dehydrogenase from a thermophile. 746 49

In renal tubules isolated from fed rabbits, 1 mM aspartate is mainly utilized for production of glutamine, glutamate, alanine, and serine, while it is not used for glucose synthesis. However, the addition of either 2 mM glycerol or 2 mM lactate, which are poor gluconeogenic substrates in renal tubules, results in acceleration of both glucose formation and incorporation of [14C]aspartate into glucose by several fold, accompanied by about a twofold decrease in glutamine synthesis and marked accumulation of glutamate and alanine. Ammonium release in renal tubules incubated with aspartate in the presence of methionine sulfoximine, an inhibitor of glutamine synthetase, is also decreased on the addition of glycerol and lactate by about two- and threefold, respectively. Since intracellular [glyceraldehyde 3-phosphate]/[3-phosphoglycerate], [glycerol 3-phosphate]/[dihydroxyacetone phosphate], [lactate]/[pyruvate], and intramitochondrial [glutamate]/[2-oxoglutarate] x [NH4+] ratios are increased in comparison with control values determined with aspartate alone, it is likely that the stimulatory effect of lactate and glycerol on glucose formation from aspartate may be due to (i) an increased availability of reducing equivalents in the cytosol resulting in an enhancement of glyceraldehyde-3-phosphate dehydrogenase activity and (ii) elevation of the mitochondrial NADH/NAD- ratio causing a decrease in glutamate dehydrogenase activity resulting in a diminished glutamine synthesis and enhanced provision of carbon skeleton of aspartate for gluconeogenesis. Stimulation of glucose formation in the presence of 1 mM aspartate + glycerol is not related to cell volume changes. However, an increase for about 30% of intracellular water space induced by 10 mM aspartate + glycerol is accompanied by both diminished gluconeogenesis and enhanced glutamine synthesis, compared with values measured with 1 mM aspartate plus glycerol.
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PMID:Glycerol and lactate induce reciprocal changes in glucose formation and glutamine production in isolated rabbit kidney-cortex tubules incubated with aspartate. 764 77

The amino acid composition of proteins from mesophilic and extremophilic organisms is commonly assumed to reflect the mechanisms of molecular adaptation to extremes of physical conditions. In this context, halophilic behaviour has been attributed to significantly increased numbers of aspartic and glutamic acid residues. However, extending the analysis to a statistically relevant set of related proteins, dihydrofolate reductase from Halobacterium volcanii, as an example, shows that the increase in negative charge is found to be less significant than other exchanges of amino acids (e.g., Ala, Asn, Arg, Lys, Phe, Ser). Thus, the high water binding capacity of negatively charged residues cannot be unambiguously correlated with the anomalous stability of halophilic proteins. A similar caveat holds for generalizations regarding the thermal stability of proteins. In this case, D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was compared with a number of mesophilic and moderately thermophilic homologs. Again, 'traffic rules of stabilization', in terms of amino acid changes in going from mesophilic to thermophilic proteins, cannot be given.
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PMID:Relevance of sequence statistics for the properties of extremophilic proteins. 790 11


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