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At least 19 recordsLinked to original sources

PubMed1977

Fatty acid synthetase from Brevibacterium ammoniagenes: formation of monounsaturated fatty acids by a multienzyme complex.

A multienzyme fatty acid synthetase complex isolated from Brevibacterium ammoniagenes has been purified to a specific activity of 1440 nmol of malonyl-CoA incorporated per min/mg. The enzyme is homogeneous, as judged by gel electrophoresis on agarose gels, and has a molecular weight of 1.2 X 10(6). Both NADPH and NADH are required for activity. In contrast to other fatty acid synthetase complexes, the enzyme catalyzes the synthesis of both long-chain saturated and monounsaturated fatty acids from malonyl-CoA and acetyl-CoA. The formation of unsaturated fatty acids is oxygen-independent and sharply reduced by 3-decynoyl-N-acetylcysteamine, a known inhibitor of Escherchia coli beta-hydroxydecanoyl thioester dehydrase (EC 4.2.1.60).

Aerobiosis↗

PubMed1992

Atomic structure of the cubic core of the pyruvate dehydrogenase multienzyme complex.

The highly symmetric pyruvate dehydrogenase multienzyme complexes have molecular masses ranging from 5 to 10 million daltons. They consist of numerous copies of three different enzymes: pyruvate dehydrogenase, dihydrolipoyl transacetylase, and lipoamide dehydrogenase. The three-dimensional crystal structure of the catalytic domain of Azotobacter vinelandii dihydrolipoyl transacetylase has been determined at 2.6 angstrom (A) resolution. Eight trimers assemble as a hollow truncated cube with an edge of 125 A, forming the core of the multienzyme complex. Coenzyme A must enter the 29 A long active site channel from the inside of the cube, and lipoamide must enter from the outside. The trimer of the catalytic domain of dihydrolipoyl transacetylase has a topology identical to chloramphenicol acetyl transferase. The atomic structure of the 24-subunit cube core provides a framework for understanding all pyruvate dehydrogenase and related multienzyme complexes.

Amino Acid Sequence↗

PubMed1976

Specific release of the thioesterase component of the fatty acid synthetase multienzyme complex by limited trypsinization.

Limited trypsinization of the fatty acid synthetase multienzyme complex from rat mammary gland results in the release of a protein, molecular weight 32,000, with thioesterase activity. The other components of the multienzyme complex--the acyl carrier protein, acetyl and malonyl transferases, condensing enzyme, keto reductase, dehydrase and enoyl reductase--are not affected and remain associated with the complex. The thioesterase can be isolated by ammonium sulfate precipitation and gel filtration. Extensive trypsinization of fatty acid synthetase multienzyme complex results in a loss of thioesterase activity, probably due to cleavage of the thioesterase component into inactive peptides. However, the molecular weight and specific activity of the thioesterase isolated after limited trypsinization is relatively unaffected by the severity of the conditions of proteolysis. Both the thioesterase and the residual trypsinized complex react with antibodies produced against the native multienzyme. The results demonstrate that mild trypsinization can be used to release the thioesterase component of the multienzyme with little perturbation of either the thioesterase or the other components of the complex.

Animals↗

PubMed1992

Biochemical and molecular genetic aspects of eukaryotic pyruvate dehydrogenase multienzyme complexes.

The alpha-keto acid dehydrogenase multienzyme complexes play central roles in metabolism, are major sites of regulation, and are clinically important. Genes and cDNAs encoding the components of these complexes have been cloned and sequenced. Protein engineering and molecular cloning experiments are providing new insight into organization, structure-function relationships, and the molecular basis of genetic defects in these multienzyme complexes.

Acyltransferases↗

PubMed1976

Fluorescence energy-transfer measurements between coenzyme A and flavin adenine dinucleotide binding sites of the Escherichia coli pyruvate dehydrogenase multienzyme complex.

The interaction of the pyruvate dehydrogenase multienzyme complex from Escherichia coli with 1,N6-etheno-CoA (epsilonCoA) and coenzyme A (CoA) has been investigated using equilibrium binding, steady-state fluorescence, and fluorescence lifetime measurements. A procedure for the resolution of the pyruvate dehydrogenase multienzyme complex into the pyruvate dehydrogenase enzyme and the transacetylase-flavoprotein subcomplex also is given. Direct binding studies with epsilonCoA indicate that 25 bound epsilonCoA molecules/multienzyme complex can be readily displaced by CoA, while approximately 21 bound epsilonCoA molecules/transacetylase-flavoprotein subcomplex can be displaced by CoA. The dissociation constant for the CoA displaceable epsilonCoA is 57.8 muM for the complex and 126 muM for the subcomplex in 0.02 M potassium phosphate (pH 7.0) at 5 degrees C. The kinetic behavior of epsilonCoA as a substrate was investigated and compared with that of CoA under a variety of conditions; the apparent Michaelis constants for epsilonCoA are considerably larger than those for CoA, while the corresponding maximal velocities are smaller. Fluorescence energy transfer measurements between bound epsilonCoA on the dihydrolipoyl transacetylase enzyme and flavin adenine dinucleotide on the dihydrolipoyl dehydrogenase enzyme either in the complex or subcomplex indicate, assuming the emission and absorption dipoles are randomly oriented, that these two probes must be at least 50 A apart.

Apoenzymes↗

PubMed1975

Dissociation and characterization of enzymes from a multienzyme complex involved in CO2 fixation.

A multienzyme complex from Euglena, molecular weight about 360,000, containing phosphoenolpyruvate carboxylase, malate dehydrogenase, and acetyl-coenzyme A carboxylase has been dissociated into active constituent enzymes. The respective molecular weights are 183,000, 67,000, and 127,000. The malate dehydrogenase contained in the complex is electrophoretically distinct from other malate dehydrogenase isozymes found in Euglena. The K-m for HCO3minus of the free and complexed acetyl-CoA carboxylase is 4.2-5.4 mM, and the substrate dependency for acetyl-CoA describes a sigmoidal relationship. The HCO3minus K-m for the free phosphoenolpyruvate carboxylase is 7.3-5.4 mM while that for the same enzyme contained in the complex is 0.7-1.3 mM. Both the free and complexed forms ofphosphoenolpyruvate carboxylase have a K-m for phosphoenolpyruvate of 0.9-1.7 mM. The latter enzyme in both the complex and free forms is stimulated by NADH, acetyl-CoA, and ATP. In the free phosphoenolpyruvate carboxylase, the stimulation passes through a maximum depending on effector concentration. The effect of NADH is to increase V-max while K-m values remain unmodified.

Acetyl Coenzyme A↗

PubMed1975

A multienzyme complex for CO2 fixation.

Acetyl-coenzyme A carboxylase from Euglena gracilis strain Z was isolated as a component of a multienzyme complex which includes phosphoenolpyruvate carboxylase and malate dehydrogenase. The multienzyme complex was shown to exist in crude extracts and was purified to a homogeneous protein with a molecular weight of 360,000 by gel filtration. The ratio of the activities of the constituent enzymes was acetyl-CoA carboxylase:phosphoenolpyruvate carboxylase:malate dehydrogenase, 1:25:500. The complex is proposed to operate in conjunction with malic enzyme, which is present in Euglena, to facilitate the formation of substrates, malonyl-CoA, and NADPH, for fatty acid biosynthesis. The interaction of the enzymes may represent a means of control of acetyl-CoA carboxylase activity in organisms which do not possess an enzyme subject to allosteric regulation. The acetyl-CoA carboxylase activity from Euglena is unaffected by citrate and isocitrate.

Acetyl-CoA Carboxylase↗

PubMed1978

Regulation of brain pyruvate dehydrogenase multienzyme complex.

A number of excellent and comprehensive reviews on various aspects of pyruvate dehydrogenase multienzyme complex have been written recently. The purpose of the present review is to summarize briefly the reaction mechanism and the regulation of this enzyme. Emphasis is put on the most recent literature not covered by previous reviews. Particular attention is also paid to the regulation of brain pyruvate dehydrogenase multienzyme complex, since a number of patients with neuromuscular diseases, such as Friedreich's ataxia, show a decreased rate of pyruvate oxidation.

Animals↗

PubMed1979

Inactivation and disassembly of the pyruvate dehydrogenase multienzyme complex from bovine kidney by limited proteolysis with an enzyme from rat liver.

Mammalian pyruvate dehydrogenase multienzyme complex is inactivated when treated with a leupeptin-sensitive enzyme (termed 'inactivase') obtained from rat liver lysosomes. However, the inactivation of the overall reaction does not affect any of the component activities of the enzyme complex. By several methods it is demonstrated that treatment with the inactivase provokes the disassembly of the complex into its constituent enzyme components which, though being enzymatically active when assayed separately, are unable to catalyze the coordinated reaction sequence of pyruvate oxidation. The dissociation occurs as a consequence of limited proteolysis of the lipoate acetyltransferase core of the multienzyme complex. Isolated nicked acetyltransferase retains its complete enzymatic activity and behaves as a high-molecular-weight aggregate. The lipoamide dehydrogenase and pyruvate dehydrogenase components, however, are not cleaved by the inactivase.

Animals↗

PubMed1976

Fluorescence energy transfer measurements between ligand binding sites of the pyruvate dehydrogenase multienzyme complex.

The interaction of the pyruvate dehydrogenase multienzyme complex from Escherichia coli with 8-anilino-1-naphthalenesulfonate (ANS), pyruvate, and acetyl-CoA has been investigated using equilibrium binding, steady-state fluorescence, and fluorescence lifetime measurements. The fluorescnece of ANS is greatly enhanced when bound to the enzyme complex and to the pyruvate dehydrogenase component of the complex. Approximately 22 molecules of ANS are bound to a molecule of the complex with a binding constant of 3.69 muM in 0.03 M potassium potassium phosphate (pH 7.0). Direct and competitive binding measurements indicate that about 42 pyruvate binding sites are present per mole of enzyme complex which has been stripped of thiamine diphosphate; the number of binding sites is reduced to 28,5 in the presence of a saturating concentration of thiochrome diphosphate, a thiamine diphosphate analogue. The dissociation constant for pyruvate to the enzyme complex in the presence of thiochrome diphosphate is 308 muM in 0.02 M potassium phosphate (pH 7.0). Pyruvate, thiochrome diphosphate, and acetyl-CoA all displace ANS from the enzyme complex. In the cases of pyruvate and thiochrome diphosphate, the concentration dependence of the displacements suggests the displacement is allosteric, while in the case of acetyl-CoA direct competition appears to be involved. GTP decreased the effect of acetyl-CoA to the enzyme complex indicate that 24-26 bound acetyl-CoA molecules per complex can be readily displaced by ANS, and the binding of acetyl-CoA to these sites displays positive cooperativity. Fluorescence energy transfer measurements between bound ANS on the pyruvate dehydrogenase enzyme and FAD on the dihydrolipoyl dehydrogenase enzyme indicate, assuming the emission and absorption dipoles are randomly oriented, that these two probes must be at least 58 A apart in the intact complex.

Anilino Naphthalenesulfonates↗

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↗

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↗

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↗

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↗

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↗

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↗