[Dissociation and reconstitution of the stable multienzyme complex fatty acid synthetase from yeast].
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Electrophoresis, Disc↗
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Purification and characterization of a multienzyme complex in the tryptophan pathway of Neurospora crassa.
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Amino Acids↗
Pyruvate dehydrogenase multienzyme complex. Characterization of assembly intermediates by sedimentation velocity analysis.
The pyruvate dehydrogenase complex is a large, highly organized assembly of several different catalytic and regulatory component enzymes. The structural core of the complex is the E2-X subcomplex, consisting of 60 dihydrolipoamide transacetylase (E2) subunits arranged in a pentagonal dodecahedron; 6 protein X and 2 pyruvate dehydrogenase kinase molecules are tightly associated with this E2 60-mer. The native E2-X subcomplex exhibits a sedimentation coefficient of 32 S. The effects of several chaotropes (guanidinium chloride, potassium thiocyanide, and urea) on the E2-X subcomplex were assessed. Treatment of the E2-X subcomplex with 4 M guanidinium chloride caused a complete loss of enzymatic activity and the dissociation of the subcomplex into monomeric 1.5-3 S species. Removal of the chaotrope by dialysis for 18 h resulted in complete restoration of E2 enzymatic activity and reassembly of a 32 S subcomplex; this reassembled subcomplex contained less protein X than the native subcomplex. Sedimentation velocity analysis of reassembled E2-X subcomplex demonstrated the presence of an 8 S assembly intermediate; this sedimentation coefficient is characteristic of globular proteins of molecular weights similar to that expected for a trimer of E2. Shorter periods of dialysis also gave rise to the 8 S species; the amount of this intermediate decreased with increasing times of dialysis. The 8 S species associated non-cooperatively to yield additional assembly intermediates exhibiting sedimentation coefficients of 10-32 S.
Acetyltransferases↗
Plasmodium falciparum possesses organelle-specific alpha-keto acid dehydrogenase complexes and lipoylation pathways.
The human malaria parasite Plasmodium falciparum possesses a single mitochondrion and a plastid-like organelle called the apicoplast. Both organelles contain members of the KADH (alpha-keto acid dehydrogenase) complexes--multienzyme complexes that are involved in intermediate metabolism. In the asexual blood stage forms of the parasites, the alpha-ketoglutarate dehydrogenase and branched chain KADH complexes are both located in the mitochondrion, whereas the pyruvate dehydrogenase is exclusively found in the apicoplast. In agreement with this distribution, Plasmodium parasites have two separate and organelle-specific pathways that guarantee lipoylation of the KADH complexes in both organelles. A biosynthetic pathway comprised of lipoic acid synthase and lipoyl (octanoyl)-ACP:protein Nepsilon-lipoyltransferase B is present in the apicoplast, whereas the mitochondrion is supplied with exogenous lipoic acid, and ligation of the metabolite to the KADH complexes is accomplished by a lipoate protein ligase A similar to that of bacteria and plants. Both pathways are excellent potential targets for the design of new antimalarial drugs.
3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
Molecular weights of mitochondrial and cytoplasmic aminoacyl-tRNA synthetases of beef liver and their complexes.
In eukaryotes, multienzyme complexes containing five to nine aminoacyl-tRNA synthetase activities have frequently been reported. In this study, we report the existence, in bovine liver cytoplasm, of a multienzyme complex containing at least 16 activities which can be disrupted by homogenization to give rise to smaller complexes and noncomplexed synthetases. Determination of the size and component activity of these complexes and of the molecular weights of all 20 free synthetases suggests that the smaller complexes and free activities normally identified arise from the larger complex by well-defined stages during homogenization. We also show that similar, though not identical, complexes are found in bovine liver mitochondria and give the molecular weights of 16 mitochondrial synthetases.
Amino Acyl-tRNA Synthetases↗
Molecular and biochemical basis of intermediate maple syrup urine disease. Occurrence of homozygous G245R and F364C mutations at the E1 alpha locus of Hispanic-Mexican patients.
Maple syrup urine disease (MSUD) is caused by a deficiency of the mitochondrial branched-chain alpha-keta acid dehydrogenase (BCKAD) complex. The multienzyme complex comprises five enzyme components, including the E1 decarboxylase with a heterotetrameric (alpha 2 beta 2) structure. Four unrelated Hispanic-Mexican MSUD patients with the intermediate clinical phenotype were diagnosed 7 to 22 mo after birth during evaluation for developmental delay. Three of the four patients were found homozygous for G to A transition at base 895 (exon 7) of the E1 alpha locus, which changes Gly-245 to Arg (G245R) in that subunit. The remaining patient was homozygous for T to G transversion at base 1,253 in the E1 alpha gene, which converts Phe-364 to Cys (F364C) in the gene product. Transfection studies in E1 alpha-deficient lymphoblasts indicate that both G245R and F364C mutant E1 alpha subunits were unable to significantly reconstitute BCKAD activity. Western blotting showed that both mutant E1 alpha subunits in transfected cells failed to efficiently rescue the normal E1 beta through assembly. The putative assembly defect was confirmed by pulse-chase labeling of E1 subunits in a chaperone-augmented bacterial overexpression system. The kinetics of initial assembly of the G245R E1 alpha subunit with the normal E1 beta was shown to be slower than the normal E1 alpha. No detectable assembly of the F364C E1 alpha with normal E1 beta was observed during the 2 h chase. Small amounts of recombinant mutant E1 proteins were produced after 15 h induction with isopropyl thiogalactoside and exhibited very low or no E1 activity. Our study establishes that G245R and F364C mutations in the E1 alpha subunit disrupt both the E1 heterotetrameric assembly and function of the BCKAD complex. Moreover, the results suggest that the G245R mutant E1 alpha allele may be important in the Hispanic-Mexican population.
3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
Regional assignment of two genes of the human branched-chain alpha-keto acid dehydrogenase complex: the E1 beta gene (BCKDHB) to chromosome 6p21-22 and the E2 gene (DBT) to chromosome 1p31.
Maple syrup urine disease (MSUD) is caused by the deficiency of the mitochondrial branched-chain alpha-keto acid dehydrogenase complex. The multienzyme complex is a macromolecule (Mr 4 X 10(6] consisting of at least six distinct subunits. In this study, the human E1 beta gene (BCKDHB) has been localized to human chromosome 6 by hybrid somatic cell analysis, and regionally assigned to chromosome bands 6p21-22 by in situ hybridization. The E2 gene (DBT), which was previously localized to chromosome 1, is regionally assigned to the chromosome band 1p31 also by in situ hybridization. Localization of the E1 beta gene to chromosome 6p21-22 assigns another major human disease locus to a region that contains several important genes, including the major histocompatability complex, tumor necrosis factor, and heat-shock protein HSP70. Mapping of the E1 beta and the E2 genes may provide information for the linkage analysis of MSUD families with mutations in these two loci.
3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
Supramolecular organization of glycolytic enzymes.
On the basis of the analysis of the data on adsorption of glycolytic enzymes to structural proteins of skeletal muscles and to the erythrocyte membranes, the data on enzyme-enzyme interactions and the data on the regulation of activity of glycolytic enzymes by cellular metabolites, the structure of the glycolytic enzymes complex adsorbed to a biological support has been proposed. The key role in the formation of multienzyme complex belongs to 6-phosphofructokinase. The enzyme molecule has two association sites, one of which provides the fixation of 6-phosphofructokinase on the support and another is saturated by fructose-1,6-bisphosphate aldolase. The multienzyme complex contains one tetrameric molecule of 6-phosphofructokinase and two molecules of each of other glycolytic enzymes. Hexokinase is not a part of the complex. The molecular mass of the multienzyme complex is about 2.6 X 10(6) daltons. The multienzyme complex has symmetry axis of second order. The formation of the multienzyme complex leads to the compartmentation of glycolytic process. The problem of integration of physico-chemical mechanisms of enzyme activity regulation (allosteric, dissociative and adsorptive mechanisms) is discussed.
Allosteric Regulation↗
[Supramolecular organization of glycolytic enzymes].
On the basis of the analysis of the data on adsorption of glycolytic enzymes to structural proteins of skeletal muscle and to erythrocyte membranes, the data on enzyme-enzyme interactions and the data on the regulation of activity of glycolytic enzymes by cellular metabolites the structure of glycolytic enzyme complex adsorbed to a biological support has been proposed. The key role in the formation of the multienzyme complex belongs to 6-phosphofructokinase. The enzyme molecule has two association sites, one of which provides the fixation of 6-phosphofructokinase on the support and another is saturated by fructose-1,6-bisphosphate aldolase. The multienzyme complex fixed on structural proteins of skeletal muscle contains one tetrameric molecule of 6-phosphofructokinase and at two molecules of other glycolytic enzymes. Hexokinase is not involved in the complex composition. The molecular mass of the multienzyme complex is about 2,6 X 10(6) Da. The formation of the multienzyme complex leads to the compartmentation of the glycolytic process. The problem of integration of physico-chemical mechanisms of enzyme activity regulation (allosteric, dissociative and adsorptive mechanisms) is discussed.
Animals↗
[Effect of composition of multienzyme cellulase complexes on rate-limiting steps for non-soluble (native) cellulose hydrolysis].
It was shown that the kinetics of multienzyme cellulase complexes are generally characterized by the absence of any single rate-limiting step of hydrolysis, even when the rates of individual steps of the process are substantially different. This peculiarity of the kinetic behaviour of cellulases can be explained in terms of certain "shunt ways" in a series of individual steps of enzymatic degradation of cellulose into glucose. A basic kinetic scheme for the degradation of cellulose was developed and proved experimentally for hydrolysis of milled cotton linters under the action of ten various cellulase complexes from the fungi Trichoderma, Geotrichum and Aspergillus. It was found that the value of the stationary rate of glucose formation in all cases is determined by the effect of two or three cellulolytic components of the complexes. It was concluded that the mechanism of native cellulose hydrolysis is the same for all cellulase complexes under study irrespective of their composition and origin.
Aspergillus niger↗
[The role of multi-enzyme complexes in the integration of cell metabolism].
General properties of enzymes and structurally ordered multienzyme complexes as controllable systems are discussed: the spatial isolation of working sites and sites of control and the realization of control mechanisms with the participation of "external" factors which provide the optimal functioning of the controllable system in the biological system of higher level of complexity. The basic mechanisms of the control of soluble enzymes are isosteric and allosteric mechanisms which directed to the maintenance of cellular homeostasis. The mechanism of functioning of a multienzyme complex as a whole which is realized with the participation of second messengers is classified as a mechanism for tracing of the signals from higher levels of the control of metabolism (from nervous, hormonal and immune systems). When discussing the control of functioning of the multienzyme complexes, special attention was paid to the complex of glycolytic enzymes formed on the structural proteins of skeletal muscles and on the membranes. An order of assembly of the complex of glycolytic enzymes is proposed. The possible localization of this complex in myofibrils is discussed.
Allosteric Regulation↗
[Biosynthesis of peptides: a non-ribosomal system].
The biosynthesis of peptides in nonribosomal systems is accomplished by complex multienzymes. These multienzymes assemble the required template for the construction of each natural product in the form of linearly coupled modules. This organization principle permits the integration of multistep synthetic processes on a single macromolecule.
Amino Acid Sequence↗
The alpha-ketoacid dehydrogenase complexes. Sequence similarity of rat pyruvate dehydrogenase with Escherichia coli and Azotobacter vinelandii alpha-ketoglutarate dehydrogenase.
The pyruvate dehydrogenase complex and the alpha-ketoglutarate dehydrogenase complex are multienzyme complexes consisting of three different enzymes. No significant similarity has been reported among the dehydrogenases which are component enzymes of these complexes, despite the presence of homology among the other component enzymes. Here we isolated cDNAs for the alpha and beta subunits of rat pyruvate dehydrogenase and they exhibited a significant similarity of the amino acid sequences among rat pyruvate dehydrogenase, 2-oxoisovalerate dehydrogenase (which is a dehydrogenase component of branched chain alpha-ketoacid dehydrogenase complex) and alpha-ketoglutarate dehydrogenase, suggesting that they have been derived from a common ancestral dehydrogenase. Our results suggested that the alpha and beta subunits of the pyruvate and 2-oxoisovalerate dehydrogenases have been derived by the cleavage of the alpha-ketoglutarate dehydrogenase. However, we could not find significant homology between rat pyruvate dehydrogenase and Gram-negative bacterial pyruvate dehydrogenase.
Amino Acid Sequence↗
Structure, assembly and function of mammalian alpha-keto acid dehydrogenase complexes.
An attempt has been made to provide a description of the mammalian alpha-keto acid dehydrogenase multienzyme complexes, especially with respect to the structure, assembly and function of PDC and OGDC based on the investigation carried out in this laboratory over the last 14 years. Special efforts have been focused on the purification, characterization, and stoichiometric resolution into component enzymes, and the reassembling process to produce these multienzyme complexes. Studies of the macroorganization and the mechanism of regulation of these multienzyme complexes still remains a most challenging question.
Acetyltransferases↗
Coenzyme A-synthesizing protein complex of Saccharomyces cerevisiae.
The coenzyme A-synthesizing protein complex (CoA-SPC) is a multienzyme complex of Saccharomyces cerevisiae (Bakers' yeast), which has a molecular weight in excess of 200,000 as determined by Sephadex G-200 column chromatography. This multienzyme complex, which is insoluble in the crude yeast cell lysate, has been purified 229-fold. A cellular component of the yeast cell lysate, referred to as t-Factor, with a molecular weight of 400-1000 and chloride ion are involved in the solubilization of CoA-SPC. The CoA-SPC requires L-cysteine, D-pantothenic acid and ATP as substrates. The terminal CoA-SPC-bound intermediate is dephospho-CoA, which is subsequently phosphorylated and released from the complex as CoA. The sequence of reactions for the synthesis of CoA by the CoA-SPC differs significantly from those previously proposed for other systems. It could be that the reaction sequence is unique for the yeast cell.
Adenosine Triphosphate↗
Absence of branched chain acyl-transferase as a cause of maple syrup urine disease.
Decreased function of human mitochondrial branched chain alpha-ketoacid dehydrogenase complex results in branched chain ketoacidemia or maple syrup urine disease. Activity of this multienzyme complex varies from 0 to approximately 15% of wild type branched chain alpha-ketoacid dehydrogenase complex activity within the population of homozygous affected individuals. We used the technique of Western Blotting with antibodies against purified bovine liver branched chain alpha-ketoacid dehydrogenase complex to screen mitochondrial proteins from cultured human fibroblasts for immunocrossreactive proteins. This method probes the physical structure of the proteins forming this multienzyme complex. One patient with branched chain ketoacidemia lacked an immunoreactive transacylase protein. This protein catalyzes the transfer of the branched chain acyl group from the decarboxylase to reduced coenzyme A. Kinetic analysis of the enzyme activity in cell lysates from this patient confirmed that the complex would not utilize coenzyme A. Thus, we have defined a structural basis for an impaired multienzyme complex of mitochondria in man.
3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
Fluorescence polarization study of the alpha-ketoglutarate dehydrogenase complex from Escherichia coli.
The lipoic acids of the alpha-ketoglutarate dehydrogenase multienzyme complex from Escherichia coli have been modified with two fluorescent probes, N-(1-pyrenyl)-maleimide and 5-[[[(iodoacetyl)amino]ethyl]amino]-naphthylene-1-sulfonic acid. Time-resolved fluorescence polarization of partially labeled complexes (18-77% inhibition of enzyme activity) reveals a complex depolarization process: one component of the anisotropy is characterized by a rotational correlation time much longer than the time scale of the measurements (less than or equal to 400 ns), reflecting the overall rotation of the complex, while a second component of the anisotropy decays with a rotational correlation time of 320 (+/- 50) ns. This decay is essentially independent of viscosity and is consistent with a model in which the depolarization is due to the dissociation from and rotation of lipoic acids between binding sites on the multienzyme complex. The sum of the rate constants characterizing the association and dissociation with the binding sites is approximately 3 x 10(6) s-1. In addition, approximately 5% of the anisotropy of the N-(1-pyrenyl)maleimide-labeled complex decays with a rotational correlation time of 25 ns; this can be attributed to local motion of the probe. At high extents of N-(1-pyrenyl)maleimide labeling (90-95% inhibition of enzyme activity), the anisotropy decay can be described by a constant term plus a rotational correlation time of about 1 microseconds. The increase in the correlation time probably reflects interactions between pyrene moieties. The N-(1-pyrenyl)maleimide-labeled dihydrolipoyl transsuccinylase core of the multienzyme complex has been isolated, and the anisotropy is constant over the observed time range of 300 ns. This suggests that the native structure is necessary for observation of lipoic acid movement within the complex. Fluorescent-labeled limited trypsin digestion fragments of the alpha-ketoglutarate dehydrogenase complex also have been isolated, and anisotropy measurements reveal substantial mobility of the label within the fragments. The time-resolved anisotropy of FAD in the native complex and in the isolated dihydrolipoyl dehydrogenase indicates some rapid local mobility of the FAD (rotational correlation time of 12 ns) that is viscosity independent, as well as a component of the anisotropy that is constant over the 35-ns time scale of the experiments.
Escherichia coli↗
Regulatory properties of the pyruvate dehydrogenase complex of Pseudomonas aeruginosa.
The pyruvate dehydrogenase multienzyme complex of Pseudomonas aeruginosa was subjected to a steady-state kinetic analysis using the exponential model for a regulatory enzyme and a sensitive statistical fitting procedure. This showed that all the substrates, pyruvate, CoA and NAD+, exhibit cooperative kinetics towards the native multienzyme complex.
Coenzyme A↗