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PubMed1985

Substrate-induced structural changes of the pyruvate dehydrogenase multienzyme complex.

The time course of the overall reaction catalyzed by the pyruvate dehydrogenase multienzyme complex produces an unexpectedly high lag (tau = 8 S) even in the presence of saturating concentrations of its substrates. The preincubation of the pyruvate dehydrogenase complex with one of the substrates alone decreases the duration of this lag, and all the substrates of the pyruvate dehydrogenase component (E1) and dihydrolipoyl transacetylase component (E2) together (pyruvate, thiamine pyrophosphate, and CoA) result in the complete disappearance of the lag. The reduction of the dihydrolipoyl dehydrogenase component (E3) of the pyruvate dehydrogenase complex with the substrates of the complex in the absence of NAD+ produces significantly different quenching in the FAD fluorescence, and then the reduction with the substrates of E3 as dihydrolipoic acid and dithioerythritol. (The formation of FADH2 was not observed in the system.) The higher fluorescence quenching in the presence of substrates of pyruvate dehydrogenase complex compared to the effect caused by the substrates of the E3 component (dihydrolipoic acid and DTE) indicates conformational changes additionally manifested in the fluorescence properties of the enzyme complex. The substrate-induced quenching of the enzyme-bound FAD fluorescence shows biphasic kinetics. The rate constant of the slow phase is comparable with the rate constant calculated from the time duration of the lag phase observed in the overall reaction. The kinetic analysis of both intensity and anisotropy decrease of the FAD fluorescence suggests a consecutive transmittance of an all substrate-coordinated, induced conformational changes directed from the pyruvate dehydrogenase-via the lipoyl transacetylase--to the lipoyl dehydrogenase. Two simultaneous conformational effects caused by binding of the substrates can be distinguished; one of them results the fluorescence of the bound FAD to be more quenched, while the other makes the FAD more mobile. The first-order rate constants of both these conformational changes were determined. The present observations suggest that the pyruvate dehydrogenase complex exists in a partially inactive state in the absence of its substrates, and it becomes active due to conformational changes caused by the binding of its substrates.

Animals↗
PubMed1990

Primary sequence of the Escherichia coli fadBA operon, encoding the fatty acid-oxidizing multienzyme complex, indicates a high degree of homology to eucaryotic enzymes.

In Escherichia coli at least five enzyme activities required for the beta-oxidation of fatty acids are associated with a multienzyme complex composed of two subunits in alpha 2 beta 2 conformation (A. Pramanik et al., J. Bacteriol. 137:469-473, 1979). In the present work, the DNA sequence of the genes encoding these two subunits, fadB and fadA, has been determined. The direction of transcription was from fadB to fadA rather than from fadA to fadB, as suggested previously (S. K. Spratt et al., J. Bacteriol. 158:535-542, 1984). Only 10 nucleotides separated the coding sequences for the two peptides, confirming the suggestion that these genes form an operon. The peptides encoded by fadB and fadA were 729 amino acids and 387 amino acids, respectively, in length. The larger and smaller peptides had predicted molecular masses of 79,678 and 40,876 Da, respectively. Recently, the sequence of the fadA gene was published in a separate report (Yang et al., J. Biol. Chem. 265:10424-10429, 1990). In this work, most of the DNA sequence for fadA was confirmed, and 10 errors were corrected. Three of these nucleotide changes resulted in five amino acid residue changes predicted in the carboxy terminus of the fadA-encoded peptide. By comparison to other peptide sequences, the alpha subunit encoded within fadB had 31% perfect identity with the rat peroxisomal enoyl-coenzyme A:hydratase-3-hydroxyacyl-coenzyme A dehydrogenase trifunctional enzyme over the entire length of the two peptides. In agreement with the work of Yang et al., the beta subunit encoded within fadA had 35 to 45% perfect identity with five thiolase genes from different eucaryotic sources over the entire length of the peptide.

Amino Acid Sequence↗
PubMed1989

A possible origin of RNA catalysis in multienzyme complexes.

Numerous attempts have recently been made to ascribe a preeminent role to RNA enzymes in primitive life systems. A model is proposed in which coenzyme-dependent RNA enzymes were initially organized in multienzyme complexes featuring (1) the continuous attachment of substrates to CoA-like carriers, as in fatty acid synthesis; and (2) the ordering of RNA enzymes via mRNA-like instructional strands. In this format, RNA enzymes would not have been required to recognize and specifically bind soluble substrates. The enzymes in this case may have required far less complexity than contemporary protein enzymes and thus less genetic information for their synthesis. An analogy is made between the proposed scheme and the protein translation mechanism, for which it may have been an evolutionary precursor.

Catalysis↗
PubMed1979

Subunit stoichiometry and molecular weight of the pyruvate dehydrogenase multienzyme complex from Escherichia coli.

The molar ratio of the component enzymes of the pyruvate dehydrogenase multienzyme complex from Escherichia coli was found to be 1.8:1.7:1[pyruvate decarboxylase (E1):dihydrolipoyl transacetylase (E2):dihydrolipoyl dehydrogenase (E3)]. This ratio was determined by measuring the Coomassie blue staining of the constituent enzymes after sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis. The above ratio is the average of four separate experiments with two different enzyme preparations. The average molecular weights of the individual enzymes were found to be 96,000, 76,000, and 55,000 for E1, E2, and E3, respectively, by sodium dodecyl sulfate and sodium dodecyl sulfate/8 M urea polyacrylamide gel electrophoresis and by column chromatography in 6 M guanidine . HCl. The molecular weight of E2 was reduced to 33,000-36,000 after extensive reduction and alkylation with iodoacetamide. The molecular weights of the complex, E1, and E3 were found to be 4,800,000, 182,000, and 104,000, respectively, with low-angle laser light scattering. Both E1 and E3 are dimeric under the conditions employed. If octahedral symmetry is assumed for the E2 core, a polypeptide chain ratio of 24:24:12 (E1:E2:E3) is in good agreement with the measured molar ratio of component enzymes and the molecular weight of the pyruvate dehydrogenase complex.

Coloring Agents↗
PubMed1983

Dual role of a single multienzyme complex in the oxidative decarboxylation of pyruvate and branched-chain 2-oxo acids in Bacillus subtilis.

The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase activities of Bacillus subtilis were found to co-purify as a single multienzyme complex. Mutants of B. subtilis with defects in the pyruvate decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) components of the pyruvate dehydrogenase complex were correspondingly affected in branched-chain 2-oxo acid dehydrogenase complex activity. Selective inhibition of the E1 or lipoate acetyltransferase (E2) components in vitro led to parallel losses in pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex activity. The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes of B. subtilis at the very least share many structural components, and are probably one and the same. The E3 component appeared to be identical for the pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes in this organism and to be the product of a single structural gene. Long-chain branched fatty acids are thought to be essential for maintaining membrane fluidity in B. subtilis, and it was observed that the ace (pyruvate dehydrogenase complex) mutant 61142 was unable rapidly to take up acetoacetate, unlike the wild-type, indicative of a defect in membrane permeability. A single pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex can be seen as an economical means of supplying two different sets of essential metabolites.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1992

Primary structures of the genes, faoA and faoB, from Pseudomonas fragi B-0771 which encode the two subunits of the HDT multienzyme complex involved in fatty acid beta-oxidation.

Three enzyme activities involved in fatty acid beta-oxidation, i.e., those of enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-oxoacyl-CoA thiolase, are exhibited by one multienzyme complex (HDT) composed of two molecules each of two peptides in Pseudomonas fragi. Using specific antisera against the two subunits of HDT, we isolated the genes encoding the subunits of HDT and designated them "faoA" (for the alpha-subunit) and "faoB" (for the beta-subunit). Their complete nucleotide sequences were determined and it was revealed that faoA and faoB, both with individual putative S.D. sequences at suitable positions, formed a cluster, in that order. The amino acid sequences deduced from the nucleotide sequences of the two genes indicated that the alpha-subunit, encoded by faoA, is a polypeptide of 715 amino acid residues, and that the beta-subunit, encoded by faoB, consists of 390 amino acid residues lacking the first methionine of the primary product encoded by faoB. Immunoblotting of cell lysates prepared from Escherichia coli transformants carrying plasmids which possess the faoA and/or faoB gene with antisera against the subunits of HDT showed that both the faoA and faoB genes were transcribed and translated in E. coli. The overall activities of 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase were increased in the E. coli cells transformed with the plasmid possessing the faoA gene, suggesting that both the hydratase and dehydrogenase activities may be exhibited by the alpha-subunit of HDT.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1989

Kinetics and specificity of reductive acylation of lipoyl domains from 2-oxo acid dehydrogenase multienzyme complexes.

Lipoamide and a peptide, Thr-Val-Glu-Gly-Asp-Lys-Ala-Ser-Met-Glu lipoylated on the N6-amino group of the lysine residue, were tested as substrates for reductive acetylation by the pyruvate decarboxylase (E1p) component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli. The peptide has the same amino acid sequence as that surrounding the three lipoyllysine residues in the lipoate acetyltransferase (E2p) component of the native enzyme complex. Lipoamide was shown to be a very poor substrate, with a Km much higher than 4 mM and a value of kcat/Km of 1.5 M-1.s-1. Under similar conditions, the three E2p lipoyl domains, excised from the pyruvate dehydrogenase complex by treatment with Staphylococcus aureus V8 proteinase, could be reductively acetylated by E1p much more readily, with a typical Km of approximately 26 microM and a typical kcat of approximately 0.8 s-1. The value of kcat/Km for the lipoyl domains, approximately 3.0 x 10(4) M-1.s-1, is about 20,000 times higher than that for lipoamide as a substrate. This indicates the great improvement in the effectiveness of lipoic acid as a substrate for E1p that accompanies the attachment of the lipoyl group to a protein domain. The free E2o lipoyl domain was similarly found to be capable of being reductively succinylated by the 2-oxoglutarate decarboxylase (E1o) component of the 2-oxoglutarate dehydrogenase complex of E. coli. The 2-oxo acid dehydrogenase complexes are specific for their particular 2-oxo acid substrates. The specificity of the E1 components was found to extend also to the lipoyl domains.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1992

Fatal combined defects in mitochondrial multienzyme complexes in two siblings.

A female child suffering from intrauterine growth retardation was born by caesarean section at 32 weeks. In the immediate newborn period there was a metabolic acidosis but this resolved. Hypotonia, muscular weakness and poor respiratory effort were evident and the child died at 6 days of age. A previous male sibling had died at 3 months of age after similar symptoms with seizures and a dysmyelination disorder. Post-mortem examination of both children showed damage to the basal ganglia. Defects in the activities of the pyruvate dehydrogenase complex, cytochrome oxidase and succinate cytochrome c reductase were found in cultured skin fibroblasts. Similar defects were found in isolated muscle mitochondria but not in isolated liver mitochondria from the patient. Immunoblotting for cytochrome oxidase showed that the multienzyme complex was not assembled in muscle and skin fibroblast mitochondria, but was assembled in liver mitochondria. Similar results were obtained in cultured skin fibroblast mitochondria for complex I of the mitochondrial respiratory chain. This is the first occasion that multiple defects have been demonstrated both in tissue and in culture skin fibroblasts in mitochondrial respiratory chain complexes.

Abnormalities, Multiple↗
PubMedDate not supplied

Inhibitor evidence for allosteric interaction in the replitase multienzyme complex.

We have previously shown that a fraction from the nuclei of S phase (DNA-synthesizing) Chinese hamster embryo fibroblasts (CHEF/18 cells) can be obtained that has a number of the enzyme activities required for DNA biosynthesis, and can catalyse the incorporation of labelled precursors into DNA (refs 1-4, also see ref. 8). This fraction, which we have termed the 'replitase', contains spherical particles of diameter approximately 25 nm, apparently multienzyme complexes for de novo DNA biosynthesis. Here we present evidence for the functional association of one of the enzyme activities, thymidylate synthase, with several of the other enzyme activities. Hydroxyurea, novobiocin and aphidicolin, inhibitors of ribonucleotide reductase, topoisomerase and DNA polymerase alpha, respectively, all inhibit thymidylate synthase in intact S phase CHEF/18 cells, but not in their soluble extracts. We suggest that these results reflect allosteric interactions between the subunits of a multienzyme DNA-synthesizing complex, which can be modulated by the specific inhibitors of individual enzyme activities in intact cells.

Animals↗
PubMed1978

Intramolecular coupling of active sites in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The intramolecular passage of substrate between the component enzymes of the pyruvate dehydrogenase multienzyme complex of Escherichia coli was examined. A series of partly reassembled complexes, varying only in their E1 (pyruvate decarboxylase, EC 1.2.4.1) content, was incubated with pyruvate in the absence of CoA, conditions under which the lipoic acid residues covalently bound to the E2 (lipoate acetyltransferase, EC2.3.1.12) chains of the complex become reductively acetylated, and the reaction then ceases. The fraction of E2 chains thus acetylated was estimated by specific reaction of the thiol groups in the acetyl-lipoic acid moieties with N-ethyl[2,3-14C]maleimide. The simplest interpretation of the results was that a single E1 dimer is capable of catalysing the rapid acetylation of 8-12 E2 chains, in good agreement with the results of Bates, Danson, Hale, Hooper & Perham [(1977) Nature (London) 268, 313-316]. This novel functional connexion of active sites must be brought about by transacetylation reactions between lipoic acid residues of neighbouring E2 chains in the enzyme complex. There was also a slow transacylation process between the rapidly acetylated lipoic acid residues and those that did not react in the initial, faster phase. This interaction was not investigated in detail, since it is too slow to be of kinetic significance in the normal enzymic reaction.

Binding Sites↗
PubMed1980

Multienzyme complex for metabolic channeling in mammalian DNA replication.

In the DNA-synthesizing phase (S phase) of CHEF/18 Chinese hamster embryo fibroblast cells, six enzymes associated with DNA metabolism, including DNA polymerase (deoxynucleoside triphosphate:DNA deoxynucleotidyl-transferase, EC 2.7.7.7), were largely localized in the nuclear region (karyoplasts). By contrast, in quiescent and G1 phase cells these enzymatic activites were mainly absent from the nucleus and were recovered in the cytoplasmic portion (cytoplasts). These nuclear (but not cytoplasmic) enzymatic activities cosedimented rapidly on sucrose density gradients. Further, the rapidly sedimenting enzyme activities were unique to cells in S phase. An organized supramolecular structure that allows channeling of metabolites into DNA was demonstrated by kinetics of nucleotide incorporation. "Permeabilized" cells selectively channeled incorporation of ribonucleoside diphosphates into DNA in preference to deoxyribonucleoside triphosphates. Deoxyribonucleoside triphosphate incorporation occurred when ribonucleoside-diphosphate reductase (2'-deoxyribonucleoside-diphosphate: oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) activity was abolished by hydroxyurea. Our interpretation is that during DNA replication, the nucleus contains a complex of DNA precursor-synthesizing enzymes juxtaposed with the "replication apparatus" comprising DNA polymerase, other enzymes, and structural proteins. Functional integrity of this structure is impaired when one of its essential components is inactivated. We propose the name "replitase" for this multienzyme complex for DNA replication and suggest that it incorporates precursors rapidly and efficiently. Possibly its assembly signals the initiation of the S phase of the cell cycle.

Animals↗
PubMed1976

Spin-label study of the mobility of enzyme-bound lipoic acid in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The lipoic acid residues covalently bound to the transacetylase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli were selectively modified by reaction with 4-maleimido-2,2,6,6-tetramethylpiperidino-oxyl. The electron-spin-resonance spectrum of the spin-labelled enzyme indicates that the bound nitroxide groups have high mobilities relative to the protein molecule. This physicochemical evidence is consistent with the view that the dithiolane ring of a lipoyl residue is capable of rapid migration between the active sites of the component enzymes in the catalytic mechanism.

Escherichia coli↗
PubMed1981

The role of lipoic acid residues in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

Two lipoic acid residues on each dihydrolipoamide acetyltransferase (E2) chain of the pyruvate dehydrogenase multienzyme complex of Escherichia coli were found to undergo oxidoreduction reactions with NAD+ catalysed by the lipoamide dehydrogenase component. It was observed that: (a) 2 mol of reagent/mol of E2 chain was incorporated when the complex was incubated with N-ethylmaleimide in the presence of acetyl-SCoA and NADH; (b) 4 mol of reagent/mol of E2 chain was incorporated when the complex was incubated with N-ethylmaleimide in the presence of NADH; (c) between 1 and 2 mol of acetyl groups/mol of E2 chain was incorporated when the complex was incubated with acetyl-SCoA plus NADH; (d) 2 mol of acetyl groups/mol of E2 chain was incorporated when the complex was incubated with pyruvate either before or after many catalytic turnovers through the overall reaction. There was no evidence to support the view that only half of the dihydrolipoic acid residues can be reoxidized by NAD+. However, chemical modification of lipoic acid residues with N-ethylmaleimide was shown to proceed faster than the accompanying loss of enzymic activity under all conditions tested, which indicates that not all the lipoyl groups are essential for activity. The most likely explanation for this result is an enzymic mechanism in which one lipoic acid residue can take over the function of another.

Acetyl Coenzyme A↗
PubMed1991

Sequence-specific 1H-NMR assignments and secondary structure of the lipoyl domain of the Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex.

The lipoyl domain (residues 1-85) of the lipoate-acetyltransferase polypeptide chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been subjected to detailed structural analysis by means of two-dimensional (2D) 1H-NMR spectroscopy at 400 MHz. Sequence-specific proton resonance assignments were made, but at this field strength not all of the side-chain protons could be assigned, especially from complex spin systems like those of leucine, proline and lysine residues. Measurement of short-range interproton distances identified two extensive regions of beta-sheet, each containing four anti-parallel peptide strands. The lipoyl-lysine residue (Lys42) is located in a tight turn at a corner of one sheet, the N-terminal and C-terminal residues of the domain are close together in two adjacent beta-strands in the other. The lipoylated and unlipoylated forms of the domain have almost identical spectra, indicating that there is little, if any, conformational change in the protein as a result of the post-translational modification.

Acetyltransferases↗
PubMed1985

Low immunogenicity of the common lipoamide dehydrogenase subunit (E3) of mammalian pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes.

The production of high-titre monospecific polyclonal antibodies against the purified pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart is described. The specificity of these antisera and their precise reactivities with the individual components of the complexes were examined by immunoblotting techniques. All the subunits of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes were strongly antigenic, with the exception of the common lipoamide dehydrogenase component (E3). The titre of antibodies raised against E3 was, in both cases, less than 2% of that of the other subunits. Specific immunoprecipitation of the dissociated N-[3H]ethylmaleimide-labelled enzymes also revealed that E3 alone was absent from the final immune complexes. Strong cross-reactivity with the enzyme present in rat liver (BRL) and ox kidney (NBL-1) cell lines was observed when the antibody against ox heart pyruvate dehydrogenase was utilized to challenge crude subcellular extracts. The immunoblotting patterns again lacked the lipoamide dehydrogenase band, also revealing differences in the apparent Mr of the lipoate acetyltransferase subunit (E2) from ox kidney and rat liver. The additional 50 000-Mr polypeptide, previously found to be associated with the pyruvate dehydrogenase complex, was apparently not a proteolytic fragment of E2 or E3, since it could be detected as a normal component in boiled sodium dodecyl sulphate extracts of whole cells. The low immunogenicity of the lipoamide dehydrogenase polypeptide may be attributed to a high degree of conservation of its primary sequence and hence tertiary structure during evolution.

Animals↗
PubMed1975

Aggregation states and catalytic properties of the multienzyme complex catalyzing the initial steps of pyrimidine biosynthesis in rat liver.

Glutamine-dependent carbamoyl-phosphate synthetase was purified about 2100-fold from the cytosol of rat liver using 30% (v/v) dimethyl sulfoxide and 5% (w/v) glycerol as stabilizers. Throughout the purification, aspartate transcarbamylase and dihydroorotase, the second and third enzymes of pyrimidine biosynthesis, were copurified with the synthetase. These three enzymes sedimented as a single peak with a sedimentation coefficient of 27 S in sucrose gradients containing the stabilizers, indicating their existence as a multienzyme complex. The aggregation states of the complex were analyzed by sucrose gradient centrifugation under conditions approximating those used for enzymatic assay and correlated with the kinetic properties of the synthetase. In the presence of 10% glycerol and 10 mM MgATP(2-) at 18 degrees, the synthetase showed high activity and the three enzymes sedimented as a single peak with a coefficient of 25 S. The three enzymes also existed as a complex with the same coefficient when 50 muM PP-ribose-P was added in place of MgATP(2-), the sedimentation coefficient of the complex shifted to 28 S, indicating alteration in its molecular shape, rather than size. With 10% glycerol alone, the complex partially dissociated and the synthetase activity appeared in three peaks with coefficients of 26, 19, and 9 S (carbamoyl-phosphate synthetases (CPSase) a, b, and c, respectively). CPSases a, b, and c, thus obtained, were all sensitive to regulation by UTP and PP-ribose-P, but they differed MgATP(2-) (5.1, 4.8, AND 1.7 mM for CPSases a and b, and the enzyme within the original complex, respectively) and in their sensitivities to effectors. These results suggest that the aggregation may modify the catalytic and regulatory properties of the synthetase; Attempts to reassociate the components were unsuccessful.

Amidohydrolases↗