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PubMed1979

Five different enzymatic activities are associated with the multienzyme complex of fatty acid oxidation from Escherichia coli.

The purified multienzyme complex of fatty acid oxidation from Escherichia coli was found to possess 3-hydroxyacyl-coenzyme A (CoA) epimerase and cis-delta3-trans-delta2-enoyl-CoA isomerase activities in addition to the previously identified enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-ketoactyl-CoA thiolase activities. Evidence is presented in support of the proposed association of all five enzyme activities with one protein which apparently is composed of two types of subunits and which can exist in several aggregated forms. The five component enzymes of the complex were rapidly inactivated by tris(hydroxymethyl)aminomethane, whereas they remained active in the presence of potassium phosphate.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1988

Search for multienzyme complexes of DNA precursor pathways in uninfected mammalian cells and in cells infected with herpes simplex virus type I.

Confirmatory evidence for the existence of a multienzyme complex of DNA precursor pathways in mammalian cells was obtained. Using neutral sucrose gradient centrifugation of cell lysates we found that at least five enzymes involved in DNA precursor metabolism in uninfected. S-phase BHK-cell fibroblasts cosediment at a common rate, indicative of a multienzyme complex. The enzymes include DNA polymerase thymidine kinase, ribonucleotide reductase, dihydrofolate reductase, and NDP-kinase. This complex was partially, but not completely, disrupted when lysates from GO-phase cells were centrifuged. Using lysates from cells infected with herpes simplex virus (HSV) type I some of the virus-induced ribonucleotide reductase and a minor proportion of the HSV-thymidine kinase cosedimented rapidly. The virus-induced DNA polymerase sedimented independently near the middle of the gradient, in contrast to the behaviour of the host polymerase. The enzyme associations observed were disrupted by NaCl or by inclusion of ethylenediamine tetraacetic acid during the cell lysis procedure, instead of the usual EGTA. These results indicate the importance of ionic forces in maintaining the enzyme complexes. The bulk of the DNA and the RNA present in the lysates did not sediment at the same rate as the complexes, showing that the enzymes were not simply adhering nonspecifically to these polyanions. Newly synthesised radiolabeled DNA (15 min pulse with [3H]thymidine) was not preferentially associated with the enzymes, but some functional DNA was evident in the enzyme complex fraction from the uninfected S-phase cells. DNA polymerase activity in this fraction did not require, nor was it stimulated by, exogenous "activated" DNA. Added DNA primer-template was required, however, for maximal activity of the polymerase in gradient fractions derived from GO-phase cells and from HSV-infected cells. No evidence for channeling of ribonucleotide precursors into DNA of permeabilized cells (uninfected or HSV-infected) was detected. Most rCDP was incorporated into RNA. In the uninfected, S-phase cells about 10 pmol/10(6) cells/90 min of rCDP residues was incorporated into DNA compared with 120 pmol/10(6) cells/90 min when radiolabeled dCTP was used. Nonradioactive dCTP present in equimolar concentration in the incubation with labeled rCDP did not, however, diminish the incorporation of label from the ribonucleotide. In permeabilized HSV-infected cells incorporation of radiolabel from rCDP into DNA was barely detectable.

Animals↗
PubMed1992

Immunoelectron microscopy of enzymes, multienzyme complexes, and selected other oligomeric proteins.

The collective term "immunoelectron microscopy" subsumes a number of techniques in which the biological material is decorated with specific antibodies, prior to being visualized in the electron microscope. In this article, we have reviewed literature on immunoelectron microscopy that focusses on the analysis of the molecular architecture of proteins, in particular of enzymes and of multienzyme complexes. Molecular immunoelectron microscopy has been remarkably successful with multi-subunit enzymes of complex quaternary structures, and in many cases the data have been the basis for the eventual development of detailed three-dimensional molecular models. The elucidation of subunit composition and juxtaposition of a given enzyme, an important accomplishment in itself, has in turn stimulated and guided discussions on the catalytic mechanism; illustrative examples include F1 ATPase and citrate lyase, among others. Here we have chosen a variety of enzymes, multienzyme complexes, and non-enzymatic proteins to demonstrate the versatility of immunoelectron microscopy, to illustrate methodological prerequisites and limitations, and to discuss significance and implications of individual immunoelectron microscopy studies.

Animals↗
PubMed1969

In vitro formation of an active multienzyme complex in the tryptophan pathway of Neurospora crassa.

A multienzyme complex that catalyzes the anthranilate synthetase, phosphoribosylanthranilate (PRA) isomerase, and indoleglycerolphosphate (InGP) synthetase reactions was produced in vitro when extracts from a tryp-1 mutant and a tryp-2 mutant of Neurospora crassa were mixed. The sedimentation values and the molecular weights for the interacting components obtained from the mutants were estimated by sucrose gradients and by gel filtration on Sephadex columns. The component coded for by the tryp-2 gene (a-component) which is present in the tryp-1-17 mutant has a sedimentation coefficient of 4.5S and molecular weight of 70,000. The component coded for by the tryp-1 gene (i-component) which is present in the tryp-2-6 mutant has a sedimentation coefficient 7.5S and a molecular weight of 170,000. The complex formed in vitro had similar properties to those previously reported for the wild-type complex. A Q(10) value of 3 and an apparent activation energy of 18,000 cal/mole were calculated for the formation of the complex from the two components.

Centrifugation, Zonal↗
PubMed1977

The subunit structure of the arom multienzyme complex of Neurospora crassa. A possible pentafunctional polypeptide chain.

A new procedure for the purification of the arom multienzyme complex from Neurospora crassa is presented. Important factors are the inactivation of proteinases by phenylmethanesulphonyl fluoride and the use of cellulose phosphate as an affinity adsorbent. A homogeneous enzyme, with a specific shikimate dehydrogenase activity of 70 units/mg of protein, is obtained in 25% yield. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, combined with cross-linking studies using dimethyl suberimidate, suggest that the complex is composed of two subunits of molecular weight 165000. Glycerol-density-gradient centrifugation indicates a molecular weight for the intact complex of about 270000. Evidence for the effects of proteolysis, both during the preparation and on storage of the purified complex, is presented, and previous reports in the literature of the occurrence of multiple subunits are discussed in this light.

Alcohol Oxidoreductases↗
PubMed1976

Selective inactivation of the transacylase components of the 2-oxo acid dehydrogenase multienzyme complexes of Escherichia coli.

1. The reaction of the pyruvate dehydrogenase multienzyme complex of Escherichia coli with maleimides was examined. In the absence of substrates, the complex showed little or no reaction with N-ethylmaleimide. However, in the presence of pyruvate and N-ethylmaleimide, inhibition of the pyruvate dehydrogenase complex was rapid. Modification of the enzyme was restricted to the transacetylase component and the inactivation was proportional to the extent of modification. The lipoamide dehydrogenase activity of the complex was unaffected by the treatment. The simplest explanation is that the lipoyl groups on the transacetylase are reductively acetylated by following the initial stages of the normal catalytic cycle, but are thereby made susceptible to modification. Attempts to characterize the reaction product strongly support this conclusion. 2. Similarly, in the presence of N-ethylmaleimide and NADH, much of the pyruvate dehydrogenase activity was lost within seconds, whereas the lipoamide dehydrogenase activity of the complex disappeared more slowly: the initial site of the reaction with the complex was found to be in the lipoyl transacetylase component. The simplest interpretation of these experiments is that NADH reduces the covalently bound lipoyl groups on the transacetylase by means of the associated lipoamide dehydrogenase component, thereby rendering them susceptible to modification. However, the dependence of the rate and extent of inactivation on NADH concentration was complex and it proved impossible to inhibit the pyruvate dehydrogenase activity completely without unacceptable modification of the other component enzymes. 3. The catalytic reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) by NADH in the presence of the pyruvate dehydrogenase complex was demonstrated. A new mechanism for this reaction is proposed in which NADH causes reduction of the enzyme-bound lipoic acid by means of the associated lipoamide dehydrogenase component and the dihydrolipoamide is then oxidized back to the disulphide form by reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). 4. A maleimide with a relatively bulky N-substituent, N-(4-diemthylamino-3,5-dinitrophenyl)maleimide, was an effective replacement for N-ethylmaleimide in these reactions with the pyruvate dehydrogenase complex. 5. The 2-oxoglutarate dehydrogenase complex of E. coli behaved very similarly to the pyruvate dehydrogenase complex, in accord with the generally accepted mechanisms of the two enzymes. 6. The treatment of the 2-oxo acid dehydrogenase complexes with maleimides in the presence of the appropriate 2-oxo acid substrate provides a simple method for selectively inhibiting the transacylase components and for introducing reporter groups on to the lipoyl groups covalently bound to those components.

Disulfides↗
PubMed1976

Use of dimethyl suberimidate and novel periodate-cleavable bis(imido esters) to study the quaternary structure of the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

Two new symmetrical bis(imido esters), N,N'-bis(2-carboximidoethyl)tartarimide dimethyl ester dihydrochloride and N,N'-bis(2-carboximidomethyl)tartarimide dimethyl ester dihydrochloride, have been synthesized. Tests with the tetrameric enzyme, fructose diphosphate aldolase, show that these reagents closely resemble dimethyl suberimidate in their ability to cross-link protein subunits. However, identification of the cross-linked species, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, is greatly facilitated since the cross-links can be broken by a simple treatment with sodium periodate. The periodate cleavage step can be introduced between the two dimensions of a diagonal gel electrophoretic separation, the contributors to a cross-linked species then moving off the diagonal formed by uncross-linked proteins and reverting to the positions in the gel that correspond with their regenerated monomeric form. When the pyruvate dehydrogenase multienzyme complex of Escherichia coli was treated with dimethyl suberimidate or N,N'-bis(2-carboximidoethyl)tartarimide dimethyl ester dihydrochloride, cross-links rapidly formed between the subunits of the transacetylase and lipoamide dehydrogenase components. On the other hand, cross-links failed to form between the subunits of the decarboxylase component themselves, or between the decarboxylase and the other two types of subunit in the complex. Cross-linking experiments with the isolated lipoamide dehydrogenase were compatible with the accepted dimeric structure of this enzyme is free solution, whereas the isolated pyruvate decarboxylase component also failed to cross-link when treated with dimethyl suberimidate in free solution. The cross-linking experiments with the intact multienzyme complex provide evidence for the existence of the lipoamide dehydrogenase dimer in the assembled enzyme and show the need to interpret such experiments with care since, from other evidence, the pyruvate decarboxylase component is known to be bound to the transacetylase "core" of the complex.

Binding Sites↗
PubMed1990

Cloning and sequence analysis of the genes encoding the dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase components of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus.

A 2641-bp EcoRI fragment of DNA that encodes the C-terminal part of the dihydrolipoyl acetyltransferase (E2) component and the dihydrolipoamide dehydrogenase (E3) component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. A 705-bp truncated open reading frame was located at the 5'end of the insert which, together with the 588-bp truncated open reading frame at the 3' end of another EcoRI fragment of B. stearothermophilus DNA previously cloned and sequenced [Hawkins, C. F., Borges, A. & Perham, R. N. (1990) Eur. J. Biochem. 191, 337-446], was identified as the gene, pdhC, encoding the E2 polypeptide chain. Direct sequence analysis of the purified E2 chain confirmed that the two EcoRI fragments are adjoining in the B. stearothermophilus genome. The E3 gene, pdhD, begins just 4 bp downstream from the stop codon of the pdhC gene. The amino acid sequences deduced from the pdhC and pdhD genes correspond to proteins of 427 amino acids (E2, Mr 46,265) and 469 amino acids (E3, Mr 49,193), respectively. Both genes are preceded by potential ribosome-binding sites and the E3 gene is followed by a stemloop structure characteristic of rho-independent transcription terminators. The B. stearothermophilus E2 and E3 chains exhibit substantial sequence similarity with the corresponding subunits of other 2-oxo-acid dehydrogenase multienzyme complexes. The cloning and sequence analysis described here complete the description of the gene cluster (pdhA, B, C and D) which encodes the B. stearothermophilus pyruvate dehydrogenase multienzyme complex.

Acetyltransferases↗
PubMed1991

Epimerization of 3-hydroxy-4-trans-decenoyl coenzyme A by a dehydration/hydration mechanism catalyzed by the multienzyme complex of fatty acid oxidation from Escherichia coli.

The mechanism of 3-hydroxyacyl-CoA epimerase (EC 5.1.2.3), which is associated with the multienzyme complex of fatty acid oxidation from Escherichia coli, was studied with D-3-hydroxy-4-trans-decenoyl-CoA as a substrate. The E. coli complex catalyzes the rapid and direct dehydration of D-3-hydroxy-4-trans-decenoyl-CoA to 2-trans,4-trans-decadienoyl-CoA, which is slowly hydrated to L-3-hydroxy-4-trans-decenoyl-CoA. A kinetic analysis of the epimerase and its partial reactions established that epimerization of 3-hydroxyacyl-CoAs occurs solely by a dehydration/hydration mechanism. The results of a substrate competition study with L-3-hydroxy-4-trans-decenoyl-CoA and its D-isomer, together with the conclusion from a sequence analysis of the large subunit of the E. coli complex (Yang, X.-Y., Schulz, H., Elzinga, M., and Yang, S.-Y. (1991) Biochemistry 30, 6788-6795), prompt the suggestion that a single active site is responsible for the dehydration of the D- and L-isomers of 3-hydroxyacyl-CoAs.

Biotransformation↗
PubMed1985

Synthesis of diadenosine 5',5''' -P1,P4-tetraphosphate by lysyl-tRNA synthetase and a multienzyme complex of aminoacyl-tRNA synthetases from rat liver.

An 18 S multienzyme complex of aminoacyl-tRNA synthetases is found to be active in the synthesis of diadenosine-5',5'''-P1,P4-tetraphosphate (AppppA). Most of the activity is attributed to lysyl-tRNA synthetase in the complex. Free lysyl-tRNA synthetase dissociated from the synthetase complex is about 6-fold more active than the complex in AppppA synthesis, while their apparent Michaelis constants for ATP and lysine are similar. AMP, which reportedly activates AppppA synthesis (Hilderman, R.H. (1983) Biochemistry 22, 4353-4357), has no effect on AppppA synthesis. The higher activity of free Lys-tRNA synthetase is in part due to the higher stimulation of AppppA synthesis by Zn2+. These results suggest that association of aminoacyl-tRNA synthetases may affect AppppA synthesis.

Adenine Nucleotides↗
PubMed1976

Steady-state kinetic formalism applied to multienzyme complexes, oxidative phosphorylation, and interacting enzymes.

A kinetic formalism, quite generally valid for free energy transducing, steady-state, macromolecular systems in biology, is applied here to multienzyme complexes, oxidative phosphorylation, and interacting enzymes. Systems of this type, comprising several interacting subunits, each with its own discrete set of states, present no new features in principle. Hence, they may be handled by the earlier kinetic formalism without modification. However, the kinetic diagram can become quite complicated because the state of each subunit (enzyme) must be specified in order to specify any one state of the system (complex) as a whole. Cycles, forces, fluxes, free energy levels, and state probabilities are considered.

Diffusion↗
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↗
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↗