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PubMed1983

Seven mammalian aminoacyl-tRNA synthetases associated within the same complex are functionally independent.

A heterotypic multienzyme complex from sheep liver containing seven aminoacyl-tRNA synthetases specific for isoleucine, leucine, methionine, glutamine, glutamic acid, lysine and arginine was subjected to kinetic analyses to examine the possibility that association of these enzymes may impart kinetic properties which differ from those of their unassociated counterparts. The evidence obtained by two different approaches leads to the conclusion that the associated enzymes are functionally independent. Firstly, the kinetic constants of the methionyl-tRNA and lysyl-tRNA synthetase components of the complex do not differ significantly from those of their unassociated counterparts obtained after controlled proteolysis of the complex. Secondly, the methionyl-tRNA synthetase component of the complex displays identical kinetic constants, whether assayed in the presence of [14C]methionine, ATP and highly enriched tRNAMet alone, or in the additional presence of the substrates required for unlabeled aminoacyl-tRNA formation by each of the other six enzymes. Similarly, the initial rates of [14C]aminoacyl-tRNA formation catalyzed by any of the six other enzymes was unaffected by the concomitant functioning of the other aminoacyl-tRNA synthetases. The sedimentation behaviour of the aminoacyl-tRNA synthetase components of the complex under conditions prevailing in the tRNA aminoacylation assay indicates that they remain associated under these conditions. The implications of these findings on the structural organization of the enzymes within the complex are discussed.

Amino Acyl-tRNA Synthetases↗
PubMed1999

Principles of quasi-equivalence and Euclidean geometry govern the assembly of cubic and dodecahedral cores of pyruvate dehydrogenase complexes.

The pyruvate dehydrogenase multienzyme complex (Mr of 5-10 million) is assembled around a structural core formed of multiple copies of dihydrolipoyl acetyltransferase (E2p), which exhibits the shape of either a cube or a dodecahedron, depending on the source. The crystal structures of the 60-meric dihydrolipoyl acyltransferase cores of Bacillus stearothermophilus and Enterococcus faecalis pyruvate dehydrogenase complexes were determined and revealed a remarkably hollow dodecahedron with an outer diameter of approximately 237 A, 12 large openings of approximately 52 A diameter across the fivefold axes, and an inner cavity with a diameter of approximately 118 A. Comparison of cubic and dodecahedral E2p assemblies shows that combining the principles of quasi-equivalence formulated by Caspar and Klug [Caspar, D. L. & Klug, A. (1962) Cold Spring Harbor Symp. Quant. Biol. 27, 1-4] with strict Euclidean geometric considerations results in predictions of the major features of the E2p dodecahedron matching the observed features almost exactly.

Acetyltransferases↗
PubMed1990

Bioregulatory mechanisms at the level of cell organelle interactions: microspectrofluorometric in situ studies.

The spatiotemporal analysis of bioregulatory mechanisms at the level of intracellular multienzyme complexes and organelle interactions is made possible by the availability of endogenous and exogenous fluorescence probes, the development of microspectrofluorometers allowing one- and two-dimensional scans of intracellular fluorescence reactions, and the use of micromanipulatory techniques enabling the rapid alteration of metabolic states. Absorbed photons are not only a tool for quantitative evaluation of metabolic processes, they can also trigger alterations of cell membranes and functions as mediated by photosensitizer drugs. In the hierarchy of intracellular organization different levels of complexity are accessible to study, such as the regulation of multienzyme complexes and the interaction of organelle complexes. Typical applications of these methods are the investigation of drug effects (e.g., on melanoma cells), metabolic and structural alterations (e.g., in cystic fibrosis and Gaucher fibroblasts), organelle interactions in cells treated with toxic agents. The implications are relevant to biotechnology for better control of metabolite production and processing, design and testing of new drugs, understanding of drug resistance and better targeting of drugs or probes to selected intracellular sites. In addition, such in vitro methods can contribute to the provision of an alternative to "whole animal experiments" as already achieved in human and mouse fibroblasts, hepatocytes, hepatoma, Swiss 3T3 cells and other cells in culture, especially with regards to an analysis of the action of xenobiotics and drugs in cell physiology and pathology, fluorescence recovery after photobleaching, study of cytoskeleton dynamics and multiparameter probing of organelle activity during in vitro wound repair.

Animals↗
PubMed2001

Characterization of two cDNAs encoding mitochondrial lipoamide dehydrogenase from Arabidopsis.

In contrast to peas (Pisum sativum), where mitochondrial lipoamide dehydrogenase is encoded by a single gene and shared between the alpha-ketoacid dehydrogenase complexes and the Gly decarboxylase complex, Arabidopsis has two genes encoding for two mitochondrial lipoamide dehydrogenases. Northern-blot analysis revealed different levels of RNA expression for the two genes in different organs; mtLPD1 had higher RNA levels in green leaves compared with the much lower level in roots. The mRNA for mtLPD2 shows the inverse pattern. The other organs examined showed nearly equal RNA expressions for both genes. Analysis of etiolated seedlings transferred to light showed a strong induction of RNA expression for mtLPD1 but only a moderate induction of mtLPD2. Based on the organ and light-dependent expression patterns, we hypothesize that mtLPD1 encodes the protein most often associated with the Gly decarboxylase complex, and mtLPD2 encodes the protein incorporated into alpha-ketoacid dehydrogenase complexes. Due to the high level of sequence conservation between the two mtLPDs, we assume that the proteins, once in the mitochondrial matrix, are interchangeable among the different multienzyme complexes. If present at high levels, one mtLPD might substitute for the other. Supporting this hypothesis are results obtained with a T-DNA knockout mutant, mtlpd2, which shows no apparent phenotypic change under laboratory growth conditions. This indicates that mtLPD1 can substitute for mtLPD2 and associate with all these multienzyme complexes.

Amino Acid Oxidoreductases↗
PubMed1980

Bovine kidney pyruvate dehydrogenase complex. Isolation of the component enzymes after limited proteolysis with papain.

1. Bovine kidney pyruvate dehydrogenase multienzyme complex is inactivated rapidly by papain. However, none of the component activities of the complex is destroyed during inactivation of the overall reaction. 2. The core component, lipoate acetyltransferase, is cleaved by papain to give principal fragments with Mr 26,500 and 26,000 (as determined by dodecylsulfate gel electrophoresis). Much more slowly, the alpha chain of the pyruvate dehydrogenase component is attacked. 3. Fragmented lipoate acetyltransferase retains its complete enzymatic activity and remains of high molecular weight. It is unable, however, to bind the other component enzymes, pyruvate dehydrogenase and lipoamide dehydrogenase. Therefore, the multienzyme complex is disassembled when treated with papain. 4. A method is described which allows the rapid and convenient isolation of nicked lipoate acetyltransferase as well as unfragmented pyruvate dehydrogenase and lipoamide dehydrogenase from papain-treated complex under very mild conditions. The two uncleaved component enzymes have identical properties and similar specific activities as enzyme preparations obtained by other, more laborious procedures.

Acetyltransferases↗
PubMed1994

The role of the proteasome in cellular protein degradation.

Eukaryotic cells contain a major intracellular proteolytic activity known as the proteasome. The proteasome is a strongly conserved cylindrical structure of high molecular weight (650 kDa, approximately 20 S) and demonstrates multiple endopeptidase activities. The general structural, biochemical and genetic features of the proteasome are conserved from archaebacteria through yeast to humans. This structure fulfills an essential role by functioning as the proteolytic core of a 26 S multienzyme complex responsible for the energy-dependent degradation of ubiquitinated proteins. The bulk of intracellular proteolysis appears to be through the ubiquitin-dependent pathway. Incorporation of the proteasome into the 26 S multienzyme complex appears to confer both a specificity for ubiquitinated proteins as well as a means to tightly regulate proteolytic activity. Thus, one function of the proteasome is required for the degradation of either abnormal or certain regulatory proteins by the ubiquitin pathway. Proteasome subunits appear to be encoded by a related gene family as defined by extensive sequence similarities. The gene products are confined to either of two general classes: alpha-type which appear to be structural and beta-type which may be catalytic. Genes encoding at least two proteasome subunits map to the Major Histocompatibility Complex. Accumulating evidence points to the proteasome (or a specialized form) participating in the cytosolic degradation of these viral proteins upon cellular infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigen Presentation↗
PubMed2001

Evidence for two pathways of thiosulfate oxidation in Starkeya novella (formerly Thiobacillus novellus).

The pathway of thiosulfate oxidation in the facultatively chemolithotrophic, sulfur-oxidizing bacterium Starkeya novella (formerly Thiobacillus novellus) has not been established beyond doubt. Recently, isolation of the sorAB genes, which encode a soluble sulfite:cytochrome c oxidoreductase, has been reported, indicating that a thiosulfate-oxidizing pathway not involving a multienzyme complex may exist in this organism. Here we report the cloning and sequencing of the soxBCD genes from S. novella, which are closely related to the corresponding genes encoding the thiosulfate-oxidizing multienzyme complex from Paracoccus pantotrophus. These findings suggest two distinct pathways for thiosulfate oxidation in S. novella. The expression of sorAB and soxC in cells grown on thiosulfate- and/or glucose-containing media was studied by Western blot analysis. The results showed that the SorAB protein is synthesized in the presence of thiosulfate irrespective of the presence of glucose. In contrast, the SoxC protein is subject to repression by glucose; the repression, however, appears to be dependent on the relative amounts of glucose and thiosulfate present. The regulatory effects observed for the expression of sorAB are likely to be mediated by an extracytoplasmic function sigma factor encoded by the sigE gene identified upstream of sorAB.

Amino Acid Sequence↗
PubMed1994

Mapping the lipoyl groups of the pyruvate dehydrogenase complex by use of gold cluster labels and scanning transmission electron microscopy.

This paper describes the organization of lipoyl moieties within the pyruvate dehydrogenase (PDH) complex from Escherichia coli as studied in the scanning transmission electron microscope (STEM). The PDH complex is a multienzyme complex consisting of E1, pyruvate dehydrogenase, E2, dihydrolipoyl transacetylase, and E3, dihydrolipoyl dehydrogenase. The core of the complex is the cubic 24-subunit E2 component, which contains the lipoyl moieties bonded to lipoyl-bearing domains. E1 and E3 are associated along the edges (E1) and on the faces (E3) of the core. The lipoyl moieties were reduced with NADH and alkylated with a p-maleimidobenzoyl undecagold cluster complex. The gold labels were found to be bound very nearly specifically by dihydrolipoyl transacetylase (E2). Undecagold clusters were imaged directly by the STEM and also digitally mapped by radial mass analysis. The mass of the E2E3 subcomplex is about half that of the PDH complex. The PDH complex and GC-PDH are both about 420 A in diameter, as determined by radial mass analysis, and the E2E3 subcomplex and GC-E2E3 are 320 and 350 A, respectively. The outer boundary of the E2E3 subcomplex was clearly shown in STEM micrographs by the undecagold labels in GC-E2E3. Data obtained from radial mass analysis of GC-E2E3 and the unlabeled E2E3 subcomplex also showed that the size of the subcomplex is extended by the lipoyl-bearing domains surrounding the central E2 core. The capabilities of lipoyl moieties to undergo translocation over long distances through structural mobility in the lipoyl-bearing domains was confirmed by the observation that many of the lipoyl groups in E2E3 subcomplexes relax outward into space vacated by the removal of E1 during the preparation of the subcomplex from PDH complex. Radial mass analysis of the PDH complex and GC-PDH indicates that lipoyl groups are distributed over a large region of the PDH complex, extending from the central core to 170-180 A from the center of the complex, with the highest density at about 75 A from the particle centers, near the interface between E2 and the associated components E1 and E3.

Escherichia coli↗
PubMed1990

Cyclosporin synthetase. The most complex peptide synthesizing multienzyme polypeptide so far described.

Cyclosporin A and its homologues are synthesized by a single multifunctional enzyme from their precursor amino acids. Cyclosporin synthetase is a polypeptide chain with a molecular mass of approximately 800 kDa. In 3% polyacrylamide-sodium dodecyl sulfate gels it shows a single band of approximately 650 kDa, which appears to not be glycosylated. The enzyme could be purified to near-homogeneity in five steps. A 72-fold purification was obtained. All constitutive amino acids of cyclosporins are activated as thioesters via aminoadenylation by the same enzyme. Then N-methylation of the thioester-bound amino acids which are present in methylated form in the cyclosporin molecule takes place, whereby S-adenosyl-L-methionine serves as the methyl group donor. Methyltransferase activity is an integral entity of the enzyme; this could be shown by a photoaffinity labeling method. 4'-Phosphopantetheine is a prosthetic group of cyclosporin synthetase similar to other peptide and depsipeptide synthetases. Cyclosporin synthetase shows cross-reactions with monoclonal antibodies directed against enniatin synthetase.

Affinity Labels↗
PubMed2001

Leucine and its keto acid enhance the coordinated expression of genes for branched-chain amino acid catabolism in Arabidopsis under sugar starvation.

Branched-chain alpha-keto acid dehydrogenase (BCKDH), a multienzyme complex, plays a key role in branched-chain amino acid catabolism. However, it remains unclear whether expression of each subunit is coordinately regulated in plants, which should be important for the efficient assembly of subunits into a functional multienzyme complex. We show that the transcripts from the Arabidopsis E1alpha subunit gene accumulated in dark-adapted leaves and in sugar-starved suspension cells. These results are complementary to our previous report that the transcripts for the E1beta and E2 subunit genes accumulated in sugar-starved cells. Expression of the E1alpha gene is likely to be regulated by hexokinase-mediated sugar signaling, indicating that sugar plays a regulatory role in the coordinated expression of BCKDH subunit genes. Furthermore, Leu and its metabolite alpha-ketoisocaproate have synergistic effects on the enhanced expression of BCKDH subunit genes under sugar starvation. We hence suggest that branched-chain amino acids activate their own degradation pathway in sugar-starved cells through co-induction of each subunit gene of BCKDH.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1993

Replitase: a complex integrating dNTP synthesis and DNA replication.

Replitase is a multienzyme complex of mammalian cells that produces deoxynucleoside triphosphates and delivers them to the DNA polymerase activity, which also resides in the complex. Structural interactions within this complex form the basis of internal controls to keep these key biosynthetic processes efficient and in balance. The active complex is formed in the nuclear region in only the S phase of the cell cycle, when the cell's DNA is being replicated. Replitase is a member of the growing family of structured, multienzyme, biosynthetic complexes for which very similar complexes are found in prokaryotes and eukaryotes. Logically, the most basic of all biosynthetic pathways should show the efficiency and precise controls that even lesser pathways possess and, in fact, this seems to be so. In this article, we have outlined a broad range of evidence supporting the existence of the replitase complex in mammalian cells, a complex for dNTP synthesis and polymerase that exists only in the S phase and only in the nuclear region. This is consistent with localization studies in intact cells and after various forms of cell fractionation and, particularly, with experiments of incorporation of precursors into DNA in isolated complexes and S phase permeabilized cells. A most forceful argument for replitase is the existence of three striking phenomena--channeling, compartmentation, and cross-inhibition. These are very difficult, perhaps impossible, to explain without replitase; with replitase, their explanation is beautifully simple.

Animals↗
PubMed2001

Solution structure of the lipoyl domain of the chimeric dihydrolipoyl dehydrogenase P64K from Neisseria meningitidis.

The antigenic P64K protein from the pathogenic bacterium Neisseria meningitidis is found in the outer membrane of the cell, and consists of two parts: an 81-residue N-terminal region and a 482-residue C-terminal region. The amino-acid sequence of the N-terminal region is homologous with the lipoyl domains of the dihydrolipoyl acyltransferase (E2) components, and that of the C-terminal region with the dihydrolipoyl dehydrogenase (E3) components, of 2-oxo acid dehydrogenase multienzyme complexes. The two parts are separated by a long linker region, similar to the linker regions in the E2 chains of 2-oxo acid dehydrogenase complexes, and it is likely this region is conformationally flexible. A subgene encoding the P64K lipoyl domain was created and over-expressed in Escherichia coli. The product was capable of post-translational modification by the lipoate protein ligase but not aberrant modification by the biotin protein ligase of E. coli. The solution structure of the apo-domain was determined by means of heteronuclear NMR spectroscopy and found to be a flattened beta barrel composed of two four-stranded antiparallel beta sheets. The lysine residue that becomes lipoylated is in an exposed beta turn that, from a [1H]-15N heteronuclear Overhauser effect experiment, appears to enjoy substantial local motion. This structure of a lipoyl domain derived from a dihydrolipoyl dehydrogenase resembles that of lipoyl domains normally found as part of the dihydrolipoyl acyltransferase component of 2-oxo acid dehydrogenase complexes and will assist in furthering the understanding of its function in a multienzyme complex and in the membrane-bound P64K protein itself.

Amino Acid Sequence↗
PubMed1986

Regulation of arginine metabolism in Saccharomyces cerevisiae. Association of arginase and ornithine transcarbamoylase.

Association of arginase and ornithine transcarbamoylase (OTCase) has been proposed to play an essential role in the regulation of arginine metabolism in Saccharomyces cerevisiae (Wiame, J.-M. (1971) Curr. Top. Cell. Reg. 4, 1-39). In this report multienzyme complex formation is directly demonstrated in the presence of the active-site ligands for OTCase and arginase. Using equilibrium sedimentation, a dissociation constant for complex formation was determined to be 2.3 X 10(-8) M in the presence of ornithine and agmatine, active-site ligands for OTCase and arginase, respectively. A molecular stoichiometry in the complex of one molecule of OTCase to one molecule of arginase was verified using transmission electron microscopy. The dimensions of the complex were determined by negative staining and rotary and unidirectional shadowing techniques to be 102 A wide by 81 A high. These dimensions are quantitively consistent with dimensions of the individual enzymes (Duong, L. T., Eisenstein, E., Green, S. M., Ornberg, R. L., and Hensley, P. (1986) J. Biol. Chem. 261, 12807-12813). The enzymatic activity of OTCase is virtually completely inhibited when associated with arginase, reflecting the dramatic modulation of enzyme activity as a consequence of the acquisition of quaternary structure in this multienzyme complex.

Arginase↗
PubMed1981

Activities of branched-chain 2-oxo acid dehydrogenase and its components in skin fibroblasts from normal and classical-maple-syrup-urine-disease subjects.

1. Comparisons of the activity and kinetics of the branched-chain 2-oxo acid dehydrogenase in cultured skin fibroblasts from normal and classical maple-syrup-urine-disease (MSUD) subjects provide a kinetic explanation for the enzyme defect. 2. In the intact cell assays, normal fibroblasts demonstrated hyperbolic kinetics with 3-methyl-2-oxo[1-14C]butyrate as a substrate. Intact fibroblasts from four classical MSUD patients showed no decarboxylation over a substrate concentration range of 0.25 to 5.0 mM, and thiamin (4 mM) was without effect. 3. The overall reaction of the multienzyme complex was efficiently reconstituted by using a disrupted-cell system. Normals again showed typical hyperbolic kinetics at the 2-oxo acid concentrations of 0.1 to 5 mM. The Vmax. and apparent Km values were 0.10 +/- 0.02 m-unit/mg of protein and 0.05-0.1 mM respectively, with 3-methyl-2-oxobutyrate. In contrast, classical MSUD patients exhibited sigmoidal kinetics (Hill coefficient, 2.5) with activity approaching 40-60% of the normal value at 5 mM substrate. The K0.5 values from the Hill plots for MSUD patients were 4-7 mM. 4. The E1 (branched-chain 2-oxo acid decarboxylase) component of the multienzyme complex was measured in disrupted-particulate preparations. Normals again showed hyperbolic kinetics with the 2-oxo acid, whereas MSUD preparations exhibited sigmoidal kinetics with the activity of E1 strictly dependent on substrate concentration. Apparent Km or K0.5 were 0.1 and 1.0 mM for normal and MSUD subjects respectively. 5. Measurements of E2 (dihydrolipoyl transacylase) and E3 (dihydrolipoyl dehydrogenase) in MSUD preparations showed them to be in the normal range. 6. The above data suggest a defect in the E1 step of branched-chain 2-oxo acid dehydrogenase in classical MSUD patients.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1983

Hydrodynamic properties and structure of the rat liver 12 S arginyl- and lysyl-tRNA synthetase complex.

Eukaryotic aminoacyl-tRNA synthetases occur in multienzyme complexes in contrast to their prokaryotic counterparts. A core 12 S rat liver complex (Mr 290,000) was recently purified to homogeneity consisting of two polypeptides with Mr 73,000 and 65,000 identified as lysyl- and arginyl-tRNA synthetase, respectively (Dang et al. (1982) Biochemistry 21,1959-1966). Using the modified hydrodynamic theory of Kirkwood (Kirkwood, J.R. (1954) J. Polym. Sci. 12,1-14), we have determined that the model most consistent with the hydrodynamic properties of the 12 S complex is a tetrameric tetrahedral model.

Amino Acyl-tRNA Synthetases↗
PubMed2001

Inhibition of the Escherichia coli pyruvate dehydrogenase complex E1 subunit and its tyrosine 177 variants by thiamin 2-thiazolone and thiamin 2-thiothiazolone diphosphates. Evidence for reversible tight-binding inhibition.

Variants of the pyruvate dehydrogenase subunit (E1; EC ) of the Escherichia coli pyruvate dehydrogenase multienzyme complex with Y177A and Y177F substitutions were created. Both variants displayed pyruvate dehydrogenase multienzyme complex activity at levels of 11% (Y177A E1) and 7% (Y177F E1) of the parental enzyme. The K(m) values for thiamin diphosphate (ThDP) were 1.58 microm (parental E1) and 6.65 microm (Y177A E1), whereas the Y177F E1 variant was not saturated at 200 microm. According to fluorescence studies, binding of ThDP was unaffected by the Tyr(177) substitutions. The ThDP analogs thiamin 2-thiazolone diphosphate (ThTDP) and thiamin 2-thiothiazolone diphosphate (ThTTDP) behaved as tight-binding inhibitors of parental E1 (K(i) = 0.003 microm for ThTDP and K(i) = 0.064 microm for ThTTDP) and the Y177A and Y177F variants. This analysis revealed that ThTDP and ThTTDP bound to parental E1 via a two-step mechanism, but that ThTDP bound to the Y177A variant via a one-step mechanism. Binding of ThTDP was affected and that of ThTTDP was unaffected by substitutions at Tyr(177). Addition of ThDP or ThTDP to parental E1 resulted in similar CD spectral changes in the near-UV region. In contrast, binding of ThTTDP to either parental E1 or the Y177A and Y177F variants was accompanied by the appearance of a positive band at 330 nm, indicating that ThTTDP was bound in a chiral environment. In combination with x-ray structural evidence on the location of Tyr(177), the kinetic and spectroscopic data suggest that Tyr(177) has a role in stabilization of some transition state(s) in the reaction pathway, starting with the free enzyme and culminating with the first irreversible step (decarboxylation), as well as in reductive acetylation of the dihydrolipoamide acetyltransferase component.

Amino Acid Sequence↗
PubMed1994

Characterization of the metal centers of the Ni/Fe-S component of the carbon-monoxide dehydrogenase enzyme complex from Methanosarcina thermophila.

Methanosarcina thermophila contains a multienzyme complex called the carbon-monoxide dehydrogenase complex, which has been resolved into a nickel/iron-sulfur and a corrinoid/iron-sulfur component. This complex plays a central role in acetoclastic methanogenesis. The Ni/Fe-S component catalyzes CO oxidation and has been proposed to be involved in cleavage of acetyl-CoA into its methyl, carbonyl, and CoA moieties. In the work reported here, three metal centers in the Ni/Fe-S component were characterized by electron paramagnetic resonance (EPR) spectroscopy and spectroelectrochemistry and pre-steady state kinetics. Center A contains nickel and iron and forms an EPR active adduct with CO, which is called the NiFeC species. The EPR spectrum of the NiFeC species has g values of 2.059, 2.051, and 2.029 and is observable at temperatures as high as 150 K. This signal had previously been observed only in the carbon-monoxide dehydrogenase complex of M. thermophila and the acetyl-CoA synthase from acetate-producing bacteria. Incubation of the CO-reduced Ni/Fe-S component with acetyl-CoA resulted in an increase in intensity of the NiFeC signal, which supports a role for the component in the cleavage of acetyl-CoA. Generation of the NiFeC EPR signal occurs with a rate constant of 0.4 s-1, a result that demonstrates the kinetic competence of this species in the acetyl-CoA cleavage reaction but rules it out as the site of oxidation of CO to CO2. Center B is likely to be a [4Fe-4S]2+/1+ center with g values of 2.04, 1.93, and 1.89 (gav = 1.95) and a standard reduction potential (E'0) of -444 mV. At potentials less than -500 mV, another EPR signal develops that appears to originate from another state of Center B. Center C is a fast relaxing center with g values of 2.02, 1.88, and 1.71 (gav = 1.87) and an E'0 of -154 mV.

Aldehyde Oxidoreductases↗
PubMed1995

Dissociation and reassembly of the dihydrolipoyl transacetylase component of the bovine heart pyruvate dehydrogenase complex.

The catalytic activity and the state of aggregation of the dihydrolipoyl transacetylase-lipoamide dehydrogenase binding protein (E2-E3BP) subcomplex of the bovine heart pyruvate dehydrogenase multienzyme complex were investigated. Treatment of E2-E3BP with the chaotropic salts GndnCl or KSCN led to a rapid decrease in transacetylase activity which was accompanied by a loss of the native quaternary structure, as indicated by changes in the sedimentation properties of the E2-E3BP subcomplex. Reassembly or refolding of dissociated E2-E3BP was achieved for the GndnCl-treated subcomplex using a defined protocol. This reassembly procedure effectively excluded all E3BP from the reassembled oligomeric transacetylase. The reassembled oligomeric E2, free of E3BP, was unable to reconstitute the overall activity of the complex following incubation with pyruvate dehydrogenase (E1) and lipoamide dehydrogenase (E3). In binding studies using radiolabeled components it was demonstrated that the reassembled transacetylase, while retaining its capacity for reductive acetylation and its ability to bind E1, lost its ability to bind E3. The evidence presented in this study indicates that the strong association of E3BP with E2 facilitates the binding of E3, the lipoamide dehydrogenase component, and therefore may have an important role in the assembly and ultimately the catalytic activity of the pyruvate dehydrogenase multienzyme complex.

Acetyltransferases↗