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Query: UNIPROT:Q07644 (polypeptide)
 72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A simple procedure is described for the purification of the pyruvate dehydrogenase complex and dihydrolipoamide dehydrogenase from Bacillus subtilis. The method is rapid and applicable to small quantities of bacterial cells. The purified pyruvate dehydrogenase complex (s0(20),w = 73S) comprises multiple copies of four different types of polypeptide chain, with apparent Mr values of 59 500, 55 000, 42 500 and 36 000: these were identified as the polypeptide chains of the lipoate acetyltransferase (E2), dihydrolipoamide dehydrogenase (E3) and the two types of subunit of the pyruvate decarboxylase (E1) components respectively. Pyruvate dehydrogenase complexes were also purified from two ace (acetate-requiring) mutants of B. subtilis. That from mutant 61142 was found to be inactive, owing to an inactive E1 component, which was bound less tightly than wild-type E1 and was gradually lost from the E2E3 subcomplex during purification. Subunit-exchange experiments demonstrated that the E2E3 subcomplex retained full enzymic activity, suggesting that the lesion was limited to the E1 component. Mutant 61141R elaborated a functional pyruvate dehydrogenase complex, but this also contained a defective E1 component, the Km for pyruvate being raised from 0.4 mM to 4.3 mM. The E1 component rapidly dissociated from the E2E3 subcomplex at low temperature (0-4 degrees C), leaving an E2E3 subcomplex which by subunit-exchange experiments was judged to retain full enzymic activity. These ace mutants provide interesting opportunities to analyse defects in the self-assembly and catalytic activity of the pyruvate dehydrogenase complex.
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PMID:Wild-type and mutant forms of the pyruvate dehydrogenase multienzyme complex from Bacillus subtilis. 640 95

Pyruvate dehydrogenase complex was purified from rat heart. The complex showed four polypeptide bands on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, corresponding to lipoate acetyltransferase (mol.wt. 68 000), lipoamide dehydrogenase (mol.wt. 56 000), alpha-subunit (mol.wt. 41 000) and beta-subunit (mol.wt. 35 000) of pyruvate dehydrogenase. Rat heart pyruvate dehydrogenase complex was dissociated into three component enzymes and the antibodies against each component enzyme were prepared. Anti-pyruvate dehydrogenase and anti-lipoate acetyltransferase antibodies effectively precipitated pyruvate dehydrogenase complex, but an anti-lipoamide dehydrogenase antibody released lipoamide dehydrogenase from the complex and effectively precipitated lipoamide dehydrogenase. Lipoamide dehydrogenase was synthesized in a cell-free reticulocyte lysate system with total RNA from rat liver. Its translation product was detected as a putative precursor which is 3000 Da larger than the mature subunit. In cell-free translation programmed with free and membrane-bound polysomes, activity of mRNA coding for the precursor of the enzyme was much higher in free polysomes than in membrane-bound polysomes.
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PMID:Purification and immunochemical studies of pyruvate dehydrogenase complex from rat heart, and cell-free synthesis of lipoamide dehydrogenase, a component of the complex. 641 56

The elucidation of the primary structure of the Escherichia coli lipoamide dehydrogenase (EC 1.8.1.4) by sequencing the corresponding structural gene (lpd) has enabled a detailed structural comparison between lipoamide dehydrogenase and the related disulphide oxido-reductase, human erythrocyte glutathione reductase (EC 1.6.4.2). Some 28% of the amino acid residues were found to be identical and a striking degree of homology was apparent throughout the polypeptide chains. It was concluded that the two enzymes possess very similar three-dimensional structures with particularly strong conservation of residues around the FAD and NAD(P) binding sites and at the redox centres of the molecules. Significant amino acid substitutions occur in the substrate binding pocket and these include an extra 18 amino acid residues at the C terminus of lipoamide dehydrogenase. Under physiological conditions, lipoamide dehydrogenase and glutathione reductase act in opposite directions, passing reducing equivalents to NAD+ or from NADPH (respectively), and two key substitutions near the redox centre could be associated with this difference in function. This study represents the first direct structural comparison between two related enzymes that are NADP+-linked (glutathione reductase) and NAD+-linked (lipoamide dehydrogenase). The differential recognition of these two cofactors could be explained in terms of amino acid substitutions. A divergent evolutionary relationship between the two enzymes including their NAD and NADP binding domains is fully supported by this analysis.
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PMID:Structural relationship between glutathione reductase and lipoamide dehydrogenase. 654 54

The pyruvate dehydrogenase complex of Pseudomonas aeruginosa PAO was purified by affinity chromatography on ethanol-Sepharose 2B followed by sucrose density gradient centrifugation. The overall purification was 130-fold based on enzyme activity. The purified complex contained three major and one minor polypeptide components when analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. These were identified by heat treatment, limited proteolysis and peptide mapping as pyruvate dehydrogenase (El; Mr 92500), acetyltransferases (E2; major component, Mr 76000, and minor component, Mr 77800) and lipoamide dehydrogenase (E3; Mr 58000). The purified complex had a sedimentation coefficient of 48S and the specific activity for the overall reaction of the complex was 6.5 micromol substrate transformed (mg protein)-1 min-1 at the optimum pH (7.8) and 25 degrees C. The lesions in four ace mutants lacking overall pyruvate dehydrogenase complex activity were identified after partial purification of the corresponding cell-free extracts. Three strains, designated ace A mutants, lacked pyruvate dehydrogenase activity (E1 component) and one strain, and ace B mutant, lacked the activity of the acetyltransferase (E2 component).
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PMID:The pyruvate dehydrogenase complex of Pseudomonas aeruginosa PAO Purification, properties and characterization of mutants. 678 86

The 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli was treated with trypsin at pH 7.0 at 0 degrees C. Loss of the overall catalytic activity was accompanied by rapid cleavage of the lipoate succinyltransferase polypeptide chains, this apparent Mr falling from 50 000 to 36 000 as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. A slower shortening of the 2-oxoglutarate decarboxylase chains was also observed, whereas the lipoamide dehydrogenase chains were unaffected. The inactive trypsin-treated enzyme had lost the lipoic acid-containing regions of the lipoate succinyltransferase polypeptide chains, yet remained a highly assembled structure, as judged by gel filtration and electron microscopy. The lipoic acid-containing regions are therefore likely to be physically exposed in the complex, protruding from the structural core formed by the lipoate succinyltransferase component between the subunits of the other component enzymes. Proton nuclear magnetic resonance spectroscopy of the 2-oxoglutarate dehydrogenase complex revealed the existence of substantial regions of polypeptide chain with remarkable intramolecular mobility, most of which were retained after removal of the lipoic acid-containing regions by treatment of the complex with trypsin. By analogy with the comparably mobile regions of the pyruvate dehydrogenase complex of E. coli, it is likely that the highly mobile regions of polypeptide chain in the 2-oxoglutarate complex are in the lipoate succinyltransferase component and encompass the lipoyl-lysine residues. It is clear, however, that the mobility of this polypeptide chain is not restricted to the immediate vicinity of these residues.
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PMID:Limited proteolysis and proton n.m.r. spectroscopy of the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli. 680 71

1. Pyruvate dehydrogenase complex from Saccharomyces cerevisiae is similar in size (s20,w 77 S) and flavin content (1.3--1.4 nmol/mg) to the complexes from mammalian mitochondria. 2. The relative molecular masses of the constituent polypeptide chains, as determined by dodecylsulfate gel electrophoresis at different gel concentrations, were: lipoate acetyltransferase (E2), 58 000; lipoamide dehydrogenase (E3), 56 000; pyruvate dehydrogenase (E1), alpha-subunit, 45 000, and beta-subunit, 35 000. Gel chromatography in the presence of 6 M guanidine . HCl gave a value of 52 000 for E2 indicating anomalous electrophoretic migration as described for the E2 components of other pyruvate dehydrogenase complexes. Thus, the organization and subunit Mr values are similar with the mammalian complexes and virtually identical with the complexes of gram-positive bacteria but differ greatly from the pyruvate dehydrogenase complexes of gram-negative bacteria. 3. The complex was resolved into its component enzymes by the following methods. E1 was obtained by treatment of the complex with elastase followed by gel chromatography on Sepharose CL-2B using a reverse ammonium sulfate gradient for elution. E2 was isolated by gel filtration of the complex in the presence of 2 M KBr, and E3 was obtained by hydroxyapatite chromatography in 8 M urea. The isolated enzymes reassociated spontaneously to give pyruvate dehydrogenase overall activity.
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PMID:Pyruvate dehydrogenase complex from baker's yeast. 2. Molecular structure, dissociation, and implications for the origin of mitochondria. 703 Jul 41

Glutathione reductase (Mr 2 x 52 500), a flavoenzyme of known three-dimensional structure, catalyses the reduction of glutathione disulfide by NADPH. This paper describes the primary structure of the FAD-binding domain which ranges from AcAla-1 to Gly-157. The three CNBr-produced fragments (69, 10 and 80 residues) of the domain were fractionated further by enzymatic and chemical methods; isolated peptides were sequenced mainly by automatic solid-phase Edman degradation. The tryptic peptides were overlapped by chymotryptic peptides. A fragment which results from cleavage at the acid-labile bond between Asp-135 and Pro-136 supplied peptides for overlapping the CNBr-produced fragments. In addition, many peptides were ordered and overlapped by computerized comparison with a complete sequence guessed from the electron density map. With one exception the computer method and the chemical alignment gave the same results. The sequence data are discussed in the light of the secondary and tertiary structure (Schulz et al. (1978) Nature (Lond.) 273, 120--124]. The 17 N-terminal residues are not visible in the electron density map. Consequently our numbering scheme differs from that of Schulz et al. by approximately 20 residues. Acetylation of the N terminus and an unusual composition of the following residues may serve to protect the loose N-terminal section of the protein against proteolysis in situ. The four cysteinyl residues of the FAD domain are of special interest. Cys-2 at the tip of the N-terminal extension is likely to be involved in the aggregation behaviour of glutathione reductase. Cys-58 and Cys-63 (formerly Cys-41 and Cys-46) represent the enzyme's redox-active dithiol. Cys-90 with its location at the twofold axis forms a disulfide bridge with Cys-90 of the other peptide chain of the enzyme. This might be related to the fact that both peptide chains contribute to each of the two active centers. In view of the interchain disulfide bridge glutathione reductase should be regarded as a monomeric protein. The sequence of the FAD-binding domain was compared with the sequence of the NADPH-binding domain of glutathione reductase using a computer program. As discussed, the scarcity of sequence similarities does not argue against the assumption that the two nucleotide-binding domains of glutathione reductase originated by gene duplication. The pyrophosphate moiety of FAD binds to a part of the polypeptide chain which in geometric structure, in topology and in sequence resembles the phosphate loops of other nucleotide-binding proteins and of flavodoxin. Using the phosphate loop as a reference, the N-terminal sequence of five flavoproteins can be aligned. The results of Williams et al. on the sequence of lipoamide dehydrogenase (EC 1.6.4.3) and our data on glutathione reductase (EC 1.6.4.2) show clearly that these two mechanistically similar enzymes possess homologous structures.
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PMID:Glutathione reductase from human erythrocytes: amino-acid sequence of the structurally known FAD-binding domain. 703 15

The pyruvate dehydrogenase (Pyruvate:lipoamide oxidoreductase (decarboxylating and acceptor acetylating), EC 1.2.4.1) complex from Salmonella typhimurium was purified, characterized and compared to the enzyme complex from Escherichia coli. No difference could be found in the molecular weights of the native enzyme complexes or in the single polypeptide chains of the enzymes of the two organisms. Values of 100 000, 87 000 and 56 000 were obtained for the polypeptide chains of the pyruvate dehydrogenase, the dihydrolipoamide transacetylase (acetyl-CoA:dihydrolipoamide S-acetyltransferase, EC 2.3.1.12) and the dihydrolipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3) components, respectively. Complete cross-reactivity was found with antibodies directed against the pyruvate dehydrogenase complex from E. coli and electron micrographs of both enzyme complexes reveal identical structures. A high Michaelis constant for pyruvate with a Km = 6 . 10(-4) M and a somewhat weaker cooperativity as compared to the enzyme from E. coli reflect some minor differences, while the binding of the cofactor thiamine diphosphate (Km = 1 . 10(-6) M) is identical for both enzyme complexes. Reassociation to a fully active complex molecule works with equal facility between the pyruvate dehydrogenase component and a dihydrolipoamide transacetylase: dihydrolipoamide dehydrogenase subcomplex from either organism in all possible combinations.
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PMID:Purification and properties of the pyruvate dehydrogenase complex from Salmonella typhimurium and formation of hybrids with the enzyme complex from Escherichia coli. 705 36

The interaction between the pyruvate decarboxylase (E1) component and a di-domain (lipoyl domain plus peripheral subunit-binding domain) from the dihydrolipoyl acetyltransferase (E2) component of the Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex was investigated. Only 1 mol of di-domain (binding domain) was bound to 1 mol of heterotetrameric E1 (alpha 2 beta 2) and the binding was without effect on the kinetic activity of E1. Similarly, the di-domain bound to separate E1 beta subunits at a maximal polypeptide chain ratio of 1:2, but no detectable interaction was found with the E1 alpha subunit. However, addition of the monomeric E1 alpha subunit to an E1 beta-di-domain complex generated a fully functional E1 (alpha 2 beta 2)-di-domain complex, indicating that the E1 beta subunit plays the critical part in binding the E1 component to the di-domain and suggesting that no chaperonin is needed in vitro to promote the assembly of the three separate proteins. Mixing the E1 and dihydrolipoyl dehydrogenase (E3) components in the presence of di-domain revealed that E1 and E3 cannot bind simultaneously to the same molecule of di-domain, a new feature of the assembly pathway and an important factor in determining the ultimate structure of the assembled enzyme complex.
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PMID:Interaction of component enzymes with the peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus: stoichiometry and specificity in self-assembly. 770 67

Binding characteristics among three catalytic components of rat liver branched-chain 2-oxo acid dehydrogenase complex (BCKADH) were investigated by ELISA. Dihydrolipoamide dehydrogenase (E3) was bound to solid-phase dihydrolipoamide acyltransferase (E2). The binding curve was hyperbolic giving a calculated half-maximal binding concentration of 167 ng/ml for E3. Specificity of the binding of E3 to E2 was certified by a competition experiment measuring binding of biotin-labeled E3 in the presence of unlabeled E3. The decarboxylase component (E1), which is the other catalytic component of the complex, prevented the E3 binding to E2. The specific binding between E2 and E3 was verified in the opposite direction with immobilized E3. E2 also bound to solid-phase E1 in a specific manner, and addition of E3 prevented the E2 binding to E1. No binding between E1 and E3 was observed. Thus, E1 and E3 prevented each other's binding to E2, suggesting that E1 and E3 may recognize overlapped binding sites on the E2 polypeptide or that they may, at least in part, sterically interact with each other on their binding to E2. The reconstitution experiment of the complex also supported such a mutually exclusive binding of E1 and E3 to the E2 core.
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PMID:Binding properties of the components of branched-chain 2-oxo acid dehydrogenase complex on ELISA. 789 39


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