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PubMed1981

Studies on the regulation of the branched chain alpha-keto acid dehydrogenase in the perfused rat liver.

The regulation of the branched chain alpha-keto acid dehydrogenase multienzyme complex was investigated in the isolated, perfused rat liver. The metabolic flux through the branched chain alpha-keto acid dehydrogenase was monitored by measuring the production of 14CO2 from infused 1-14C-labeled branched chain alpha-keto acid substrates. The rate of decarboxylation of alpha-keto[1-14C]isocaproate exceeded that of alpha-keto[1-14C]isovalerate at all concentrations of the substrates infused. Coinfusion of either alpha-ketoisovalerate or alpha-keto-beta-methylvalerate inhibited the rate of alpha-keto[1-14C]isocaproate decarboxylation. The rate of alpha-keto[1-14C]isovalerate decarboxylation ws enhanced during coinfusion of L(--)carnitine, while alpha-keto[1-14C]isocaproate decarboxylation was unaffected. The presence of pyruvate in the perfusion medium resulted in an inhibition of the flux through the branched chain complex with either alpha-ketoisocaproate or alpha-ketoisovalerate as the substrate. DL-beta-hydroxybutyrate infusion inhibited alpha-keto[1-14C]isocaproate decarboxylation by 18% but resulted in nearly a 100% stimulation of alpha-keto[1-14C]isovalerate decarboxylation. The evidence presented indicates that (alpha) the metabolic flux through the branched chain alpha-keto acid dehydrogenase complex can be monitored effectively in a continuous fashion in the perfused liver by following the release of 14CO2 from infused 1-14C-labeled substrates and (b) the changes observed in the metabolic flux through the branched chain complex during coinfusion of alternative substrates and other compounds may be entirely different depending upon which branched chain alpha-keto acid substrate is utilized to monitor this reaction.

3-Hydroxybutyric Acid↗

PubMed1987

Reevaluation of citrate lyase from Escherichia coli.

The subunit structure of citrate lyase from Escherichia coli was shown to be similar to that of all other lyases investigated so far. The three different subunits with molecular masses of 55.5 kDa, (large subunit) 35 kDa (medium-sized subunit) and 12.5 kDa (small subunit, acyl carrier protein) occurred in a ratio of 1:1:1. Using high-pressure liquid chromatography, it was possible to demonstrate that the reported large acyl carrier protein, with a molecular mass of 85 kDa was a contaminating protein associated with citrate lyase multienzyme complex; it could be removed by anion-exchange chromatography with Q-Sepharose. The typical two configurations of citrate lyase, the 'star' form and the 'ring' form with a diameter of 14.3 nm and 15.4 nm, respectively, could be detected by electron microscopy.

Bacterial Proteins↗

PubMed2002

The 3' end formation in small RNAs.

Small RNAs are a major class of RNAs along with transfer RNAs, ribosomal RNAs, and messenger RNAs. They vary in size from less than 100 nucleotides to several thousand nucleotides and have been identified and characterized both in prokaryotes and eukaryotes. Small RNAs participate in a variety of cellular functions including regulating RNA synthesis, RNA processing, guiding modifications in RNA, and in transport of proteins. Small RNAs are generated by a series of posttranscriptional processing steps following transcription. While RNA 5' end structure, 5' cap formation, and RNA processing mechanisms have been fairly well characterized, the 3' end processing is poorly understood. Recent data point to an emerging theme in small RNAs metabolism in which the 3' end processing is mediated by the exosome, a large multienzyme complex. In addition to removal of nucleotides by the exosome, there is simultaneous rebuilding of the 3' end of some small RNA by adenylation and/or uridylation. This review presents a picture of both degradative and rebuilding reactions operative on the 3' end of some small RNA molecules in prokaryotes and eukaryotes.

Animals↗

PubMed2005

Multiple modes of active center communication in thiamin diphosphate-dependent enzymes.

Detection of interaction between cofactors at the active centers of homodimeric and homotetrameric enzymes is usually elusive by steady-state kinetic approaches and requires protein variants where such interactions are diminished or exaggerated. In this Account, evidence for active-center interactions will be presented for the following thiamin diphosphate-dependent enzymes: yeast pyruvate decarboxylase, benzoylformate decarboxylase, and examples from the 2-oxoacid dehydrogenase multienzyme complex class. The dissymmetry of active sites is especially evident in the X-ray structures of these enzymes with substrate/substrate analogues bound. Perturbations that reveal active center communication include use of chromophoric substrates and substitutions of active center residues on putative pathways.

Binding Sites↗

PubMed1979

Purification and characterization of branched chain alpha-ketoacid dehydrogenase from bovine liver mitochondria.

Branched chain alpha-ketoacid dehydrogenase (EC 1.2.4.3(4)) was solubilized and purified from bovine liver mitochondria for the first time. Decarboxylation of alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, and alpha-ketoisocaproate was catalyzed by this multienzyme complex and this activity was co-purified for each substrate. Three enzymatic functions were contained in the complex including decarboxylation of the above ketoacids, transacylation of their simple acid derivatives, and reduction of NAD+ as an overall reaction. Product stoichiometry of these three reactions was 1 CO2:1 acyl-CoA:1 NADH. Activity depended upon the addition of thiamin pyrophosphate, CoASH, and NAD+ which were dissociable cofactors. Physically, two active forms of the enzyme complex were found: a 275,000-dalton unit and a 2 x 10(6)-dalton component. Both showed a characteristic flavin spectra and catalyzed all functions of the complex, implying that 10 small units aggregated into the larger unit. The soluble complex as visualized by electron microscopy had a diameter ranging from 12 to 24 nm corresponding to a molecular weight of 2 x 10(6). The size of the native membrane-bound component remains to be determined.

Amino Acids, Branched-Chain↗

PubMed1977

Evidence for a complex of three beta-oxidation enzymes in Escherichia coli: induction and localization.

The enzymes for beta-oxidation of fatty acids in inducible and constitutive strains of Escherichia coli were assayed in soluble and membrane fractions of disrupted cells by using fatty acid and acyl-coenzyme A (CoA) substrates containing either 4 or 16 carbon atoms in the acyl moieties. Cell fractionation was monitored, using succinic dehydrogenase as a membrane marker and glucose 6-phosphate dehydrogenase as a soluble marker. Acyl-CoA synthetase activity was detected exclusively in the membrane fraction, whereas acyl-CoA dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and 3-ketoacyl-CoA thiolase activities that utilized both C4 and C16 acyl-CoA substrates were isolated from the soluble fraction. 3-Hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and 3-ketoacyl-CoA thiolase activities assayed with both C4 and C16 acyl-CoA substrates co-chromatographed on gel filtration and ion-exchange columns and cosedimented in glycerol gradients. The data show that these three enzyme activities of the fad regulon can be isolated as a multienzyme complex. This complex dissociates in very dilute preparations; however, in those preparations where the three activities are separated, the fractionated species retain activity with both C4 and C16 acyl-CoA substrates.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1993

Stable correction of maple syrup urine disease in cells from a Mennonite patient by retroviral-mediated gene transfer.

We have successfully used retroviral gene transfer to correct the deficiency of the branched-chain alpha-oxo acid dehydrogenase complex in lymphoblasts from a homozygous Mennonite maple syrup urine disease (MSUD) patient. The mutation in Mennonites is a Tyr-393 to Asn substitution in the branched-chain alpha-oxo acid decarboxylase (E1)alpha subunit of the enzyme complex. This promotes improper assembly of mutant E1 alpha with E1 beta subunits, leading to degradation of both polypeptides. For transduction studies, a full-length human E1 alpha CDNA was inserted into the retroviral vector LXSN to produce the recombinant LSN-E1 alpha. High-titre [6 x 10(5) colony-forming units/ml] amphotropic retroviral preparations free of helper viruses were obtained by co-cultivation of infected GP+E86 with PA317 cells. Transduction of MSUD lymphoblasts from the Mennonite patient with LSN-E1 alpha viruses restored the decarboxylation of alpha-oxo[1-14C]isovalerate to the normal level. The normal decarboxylation activity in transduced MSUD cells remained stable without G418 selection during the 14 weeks studied. Southern-blot analysis indicated that the recombinant E1 alpha cDNA was integrated into the host genome. Northern and Western blotting showed that both the normal E1 alpha mRNA and the subunit were properly expressed in transduced MSUD cells. However, the level of E1 beta subunits is lower than that of normal cells, suggesting competition of the recombinant E1 alpha with the mutant form for assembly with E1 beta. The results provide a paradigm for the development of somatic gene therapy for disorders involving mitochondrial multienzyme complexes.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed1992

Interactions between mitochondria and cytoplasm in isolated hepatocytes.

Results from a wide variety of metabolic studies have provided indirect support for conclusions derived from enzymological approaches that the enzymes of the so-called soluble cytoplasm (and the mitochondrial matrix) exist within the cell and function in the form of multienzyme complexes and that metabolite channeling takes place between the enzymes of each complex. Our studies support the possibility that the enzymes of glycolysis in liver are segregated from those of gluconeogenesis. Thus, the segregation and aggregation of Krebs cycle enzymes in the mitochondrial matrix, elucidated by Paul Srere, may be an example of a general pattern of enzyme organization pertaining to all metabolic pathways.

Animals↗

PubMed1994

Identification of the 100-kD victorin binding protein from oats.

The fungus Cochliobolus victoriae, the causal agent of victoria blight of oats, produces the host-specific toxin victorin. Sensitivity of oats to victorin, and thus susceptibility to the fungus, is controlled by a single dominant gene. This gene is believed to also confer resistance to the crown rust pathogen Puccinia coronata. In the case of victoria blight, the gene has been hypothesized to condition susceptibility by encoding a toxin receptor. A 100-kD victorin binding protein (VBP) has been identified; it binds radiolabeled victorin derivatives in a ligand-specific manner and in a genotype-specific manner in vivo. The VBP may function as a toxin receptor. In vitro translation coupled with indirect immunoprecipitation was used to identify the mRNA for the 100-kD VBP, and fractionated mRNAs were used to prepare cDNA libraries enriched in the relative abundance of cDNA for the 100-kD VBP. A 3.4-kb cDNA clone was isolated that, when subjected to a 400-bp 5' deletion, was capable of directing the synthesis of a protein in Escherichia coli, which reacted to an antibody specific for the 100-kD VBP. Peptide mapping, by limited proteolysis, indicated that the protein directed by the cDNA is the 100-kD VBP. Nucleotide sequence analysis of the cDNA revealed extensive homology to a previously cloned cDNA for the P protein component of the multienzyme complex glycine decarboxylase. Glycine decarboxylase is a nuclear-encoded, mitochondrial enzyme complex. Protein gel blot analysis indicated that the 100-kD VBP copurifies with mitochondria. Based on analysis of in vitro translation products, nucleotide sequence homology, mitochondrial localization, and the widespread species distribution of the 100-kD VBP, we concluded that the 100-kD VBP is the P protein component of glycine decarboxylase.

Amino Acid Oxidoreductases↗

PubMed1996

Tubulointerstitial nephritis with renal tubular acidosis and asymptomatic primary biliary cirrhosis accompanied by antibody to a 52-kDa mitochondrial protein alone.

We report a patient presenting with a mixed type of renal tubular acidosis, who demonstrated anti-mitochondrial antibodies on immunofluorescent study. However, study of anti-M2 antibody (enzyme immunoassay) was negative. Renal biopsy revealed lymphocyte infiltration in the interstitium compatible with chronic tubulointerstitial nephritis. Liver biopsy demonstrated a mild degree of primary biliary cirrhosis (PBC) but biochemical liver function tests were normal, resulting in a diagnosis of asymptomatic PBC. Using affinity chromatography conjugated with the patient's IgG, we purified a 52-kDa protein from a porcine renal mitochondrial fraction. This protein was identified as a component of a mitochondrial multienzyme complex such as dihydrolipoamide acyltransferase of the branched-chain alpha-keto acid dehydrogenase complex (BCKD), based on the molecular mass analysis and partial amino acid sequence of the purified protein. This is the first report of the detection of antibody to 52-kDa mitochondrial protein alone in a patient who showed predominantly tubulointerstitial damage in the kidney rather than liver damage.

Acidosis, Renal Tubular↗

PubMed1973

Enzyme assay in microsomes below zero degrees.

Reactions of a membrane-bound multienzyme complex (electron-transport chain of rat-liver microsomes) suspended in aqueous-organic solvent used as antifreeze at temperatures below 0 degrees were studied. In the presence of a relatively high concentration of ethylene glycol, electron transfer can still be observed and some individual and sequential enzyme assays can be performed over a wide range of temperatures below 0 degrees .

Animals↗

PubMed2000

The structural properties of plant peroxisomes and their metabolic significance.

Plant peroxisomes can be isolated by Percoll density gradient centrifugation at high purity and metabolic competence as well as in relatively large quantities. According to biochemical and electrophysiological analyses, plant peroxisomes have recently been shown to differ from other cell organelles in essential structural properties. Unlike mitochondria or plastids, compartmentalization of plant peroxisomal metabolism is in major parts not caused by a boundary function of the membrane but is primarily due to the specific structure of the protein matrix. The enzymes of the photorespiratory C2 cycle of leaf peroxisomes are arranged as multienzyme complexes that allow efficient metabolic channelling with high flux rates and minimum leakage of reactive oxygen species from the organelle. Transfer of metabolites, such as carboxylates, proceeds across the peroxisomal membrane via a porin-like channel, which represents a relatively unspecific but highly efficient transport system. Because all variants of peroxisomes, which all contain only a single boundary membrane, are confronted with the task of transporting a large group of metabolites while preventing the escape of reactive intermediates, it is reasonable to speculate that the unique compartmentalization feature of leaf peroxisomes also applies to peroxisomes from fungi and mammals.

Biological Transport↗

PubMed2005

Structural studies of MFE-1: the 1.9 A crystal structure of the dehydrogenase part of rat peroxisomal MFE-1.

The 1.9 A structure of the C-terminal dehydrogenase part of the rat peroxisomal monomeric multifunctional enzyme type 1 (MFE-1) has been determined. In this construct (residues 260-722 and referred to as MFE1-DH) the N-terminal hydratase part of MFE-1 has been deleted. The structure of MFE1-DH shows that it consists of an N-terminal helix, followed by a Rossmann-fold domain (domain C), followed by two tightly associated helical domains (domains D and E), which have similar topology. The structure of MFE1-DH is compared with the two known homologous structures: human mitochondrial 3-hydroxyacyl-CoA dehydrogenase (HAD; sequence identity is 33%) (which is dimeric and monofunctional) and with the dimeric multifunctional alpha-chain (alphaFOM; sequence identity is 28%) of the bacterial fatty acid beta-oxidation alpha2beta2-multienzyme complex. Like MFE-1, alphaFOM has an N-terminal hydratase part and a C-terminal dehydrogenase part, and the structure comparisons show that the N-terminal helix of MFE1-DH corresponds to the alphaFOM linker helix, located between its hydratase and dehydrogenase part. It is also shown that this helix corresponds to the C-terminal helix-10 of the hydratase/isomerase superfamily, suggesting that functionally it belongs to the N-terminal hydratase part of MFE-1.

Amino Acid Sequence↗

PubMedDate not supplied

[Properties of phosphoprotein phosphatase from the rat liver].

Prosphoproteid phosphatase, an enzyme highly specific to lysyl-tRNA-synthetase and proteins of the high-molecular-multienzymic complex of aminoacyl-tRNA-synthetases, was isolated from the rat liver. The data of electrophoresis in 4-30% PAAG with the presence of DS-Na have shown that phosphoproteid phosphatase is homogeneous and its molecular mass is 56 kDa. The isolated phosphoproteid phosphatase is activated by 2.5 mM Mg2+, Mn2+ and is inhibited by ions of univalent metals ions--200 mM Na+, 5 mM K+ as well as by 1 mM ATP, ADP, AMP.

Amino Acyl-tRNA Synthetases↗

PubMed1983

[Gene-enzyme relationships of the arom aggregate of Schizosaccharomyces pombe].

The gene-enzyme relationships of the arom multienzyme complex of Schizosaccharomyces pombe that catalyzes steps two through six in the prechorismate polyaromatic amino acid biosynthetic pathway have been studied. The various mutants were subjected to biochemical analysis by direct enzymic assays. These studies have established that aro-3A, aro-3B, aro-3C, aro-3D, and aro-3E mutants lack, respectively, the enzymic activities 5-dehydroquinate synthase, 5-dehydroquinase, shekimate kinase, 3-enolpyruvylshikimate 5-phosphate synthase, and shikimate: NADP oxidoreductase. In S. pombe lack enzymic activities for the inducible quinate catabolic pathway. The functional significance of the arom aggregate is discussed.

Alcohol Oxidoreductases↗

PubMed1987

Molecular cloning of genes encoding branched-chain keto acid dehydrogenase of Pseudomonas putida.

We cloned the structural genes for the individual subunits of the branched-chain keto acid dehydrogenase multienzyme complex on a 7.8-kilobase EcoRI-SstI restriction fragment of Pseudomonas putida chromosomal DNA by cloning into the broad-host-range vector pKT230. A direct selection system for growth on valine-isoleucine agar was achieved by complementation of P. putida branched-chain keto acid dehydrogenase mutants. The recombinant plasmid, pSS1-1, increased expression of branched-chain keto acid dehydrogenase up to five times in wild-type P. putida. The complex was expressed constitutively in P. putida(pSS1-1) but was inducible in Escherichia coli HB101(pSS1-1) by high valine. E. coli minicells transformed with pSS1-1 produced three polypeptides which did not match the four polypeptides of the purified complex. To resolve this problem, we inserted P. putida DNA from pSS1-1 into pUC18 and pUC19. The pUC-derived plasmids were used as DNA templates in an E. coli transcription-translation system. Four polypeptides were produced from the pUC18-derived plasmid which had the correct molecular weights, showing that the structural genes had been cloned. Since only weak bands were produced with the pUC19-derived plasmid, the direction of transcription was established. The locations and order of all the structural genes of branched-chain keto acid dehydrogenase were located by restriction enzyme mapping.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed1997

Glutaminyl-tRNA synthetase.

Among the twenty aminoacyl-tRNA synthetases glutaminyl-tRNA synthetase occupies a special position: it is one of only two enzymes of this family which is not found in all organisms, being mainly absent from gram positive eubacteria, archaebacteria and organelles. The E. coli GlnRS is relatively small with 553 amino acids and a molecular mass of 64.4 kDa and functions as a monomer. The mammalian enzymes are somewhat larger and can be parts of multienzyme complexes. Crystal structures were solved of E. coli GlnRS complexed with tRNA(Gln) and ATP, of this complex containing tRNA(Gln) replaced by unmodified tRNA(Gln), and of three complexes with mutated GlnRS enzymes. The GlnRS molecule consists of four domains, the catalytic site is located in the Rossman fold, typical for class I synthetases, and the reaction mechanism follows the normal adenylate pathway. The enzyme shows many similarities with glutamyl-tRNA synthetase; a common ancestor of both molecules is well established. In the E. coli system recognition of the cognate tRNA has been studied in many details using both natural and artificial mutants of tRNA(Gln) and of the enzyme: GlnRS recognizes mainly conventional parts of the tRNA molecule, namely some bases of the anticodon loop and parts of the acceptor stem.

Adenosine Triphosphate↗

PubMed1995

On the functional organization in a biological structure: the example of enzyme organization.

In this paper, we have considered how the spatial localization of enzymatic reactions, ranging from the elementary type (one step) to that of a metabolic pathway in 2 different phases, may affect the stability of metabolite concentrations. The spatial localization of molecules in the reactions involves: (1) the confinement of some enzymes to cellular substructures (organelles, membranes, cytoskeleton, multienzyme complexes); (2) exchanges of metabolites between cellular substructures (local phase) and cytosol. This organization may be called as structural. Under these conditions, we have studied the dynamical behaviour of the metabolic pathway investigating the velocity of convergence towards the reference steady-state after perturbation of metabolite concentrations. This type of stability may be called as functional stability. We show that an increase in exchanges by diffusion of metabolites between the local phase and cytosol from one hand, or a decrease in the local phase volume on the other hand, result in an increase of the functional stability around the steady-state. This is verified for one step of the pathway as well as for the entire pathway or when the pathway is present in the local phase and in the cytosol.

Enzyme Stability↗