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1.
T Wagenknecht R Grassucci G A Radke T E Roche 《The Journal of biological chemistry》1991,266(36):24650-24656
Cryoelectron microscopy has been performed on frozen-hydrated pyruvate dehydrogenase complexes from bovine heart and kidney and on various subcomplexes consisting of the dihydrolipoyl transacetylase-based (E2) core and substoichiometric levels of the other two major components, pyruvate dehydrogenase (E1) and dihydrolipoyl dehydrogenase (E3). The diameter of frozen-hydrated pyruvate dehydrogenase complex (PDC) is 50 nm, which is significantly larger than previously reported values. On the basis of micrographs of the subcomplexes, it is concluded that the E1 and E3 are attached to the E2-core complex by extended (4-6 nm maximally) flexible tethers. PDC constructed in this manner would probably collapse and appear smaller than its native size when dehydrated, as was the case in previous electron microscopy studies. The tether linking E1 to the core involves the hinge sequence located between the E1-binding and catalytic domains in the primary sequence of E2, whereas the tether linking E3 is probably derived from a similar hinge-type sequence in component X. Tilting of the E2-based cores and comparison with model structures confirmed that their overall shape is that of a pentagonal dodecahedron. The approximately 6 copies of protein X present in PDC do not appear to be clustered in one or two regions of the complex and are not likely to be symmetrically distributed. 相似文献
2.
The dihydrolipoyl transacetylase (E2p) component of the pyruvate dehydrogenase complex (PDC) of Escherichia coli is a multidomain polypeptide comprising a catalytic domain, a domain that binds dihydrolipoyl dehydrogenase (E3-binding domain), and three domains containing lipoic acid (lipoyl domains). In PDC 24 subunits of E2p associate by means of interactions involving the catalytic domains to form the structural core of PDC. From cryoelectron microscopy and computer image analysis of frozen-hydrated isolated E2p cores it appears that the lipoyl domains are located peripherally about the core complex and do not assume fixed positions. To further test this interpretation the visibility of the lipoyl domains in electron micrographs was enhanced by specifically biotinylating the lipoic acids and labeling them with streptavidin. In agreement with the studies of native, unlabeled E2p cores, cryoelectron microscopy of the streptavidin-labeled E2p cores showed that the lipoic acid moieties are capable of extending approximately 13 nm from the surface of the core. Localization of the E3-binding domains was accomplished by cryoelectron microscopy of E2p-E3 subcomplexes prepared by reconstitution in vitro. Frequently an apparent gap of several nanometers separated the bound E3 from the surface of the core. The third component of PDC, pyruvate dehydrogenase (E1p), appeared to bind to the E2p core in a manner similar to that observed for E3. These results support a structural model of the E2p core in which the catalytic, E3-binding, and three lipoyl domains are interconnected by linker sequences that assume extended and flexible conformations. 相似文献
3.
Low immunogenicity of the common lipoamide dehydrogenase subunit (E3) of mammalian pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes. 总被引:3,自引:0,他引:3
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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. 相似文献
4.
Amino acid sequence analysis of the lipoyl and peripheral subunit-binding domains in the lipoate acetyltransferase component of the pyruvate dehydrogenase complex from Bacillus stearothermophilus. 总被引:3,自引:0,他引:3
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The pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus comprises a structural core, composed of 60 dihydrolipoamide acetyltransferase (E2p) subunits, which binds multiple copies of pyruvate decarboxylase (E1p) and dihydrolipoamide dehydrogenase (E3) subunits. After limited proteolysis with chymotrypsin, the N-terminal lipoyl domain of E2p was excised, purified and sequenced. The residual complex, which remained assembled, was then digested with trypsin under mild conditions. This treatment promoted complete disassembly of the complex and the various components were separated by gel filtration and h.p.l.c. A folded fragment of E2p containing about 50 amino acid residues was identified as being responsible for binding the E3 subunits, although, unlike the corresponding region of the E2p or E2o chains of the pyruvate dehydrogenase or 2-oxoglutarate dehydrogenase complexes from Escherichia coli, the fragment also bound E1p molecules. Further peptide purification and sequence analysis allowed the determination of the first 211 amino acid residues of the B. stearothermophilus E2p chain, thus providing the complete primary structure of the lipoyl domain, the E1p/E3-binding domain and the regions of polypeptide chain, probably highly flexible in nature, that link the domains to each other and to the inner-core (E2p-binding) domain. Several of the proteolytically sensitive sites were also identified. The sequence of the B. stearothermophilus E2p chain shows close homology with the sequences of the E2p and E2o chains from E. coli, although significant differences in structure are apparent. Detailed evidence for the sequence of the peptides obtained by limited proteolysis and further chemical and enzymic cleavages have been deposited as Supplementary Publication SUP 50142 (11 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 6BQ, U.K., from whom copies may be obtained as indicated in Biochem. J. (1988) 249, 5. 相似文献
5.
6.
Dual role of a single multienzyme complex in the oxidative decarboxylation of pyruvate and branched-chain 2-oxo acids in Bacillus subtilis. 总被引:9,自引:0,他引:9
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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. 相似文献
7.
8.
Improvement of diffraction quality upon rehydration of dehydrated icosahedral Enterococcus faecalis pyruvate dehydrogenase core crystals.
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T. Izard S. Sarfaty A. Westphal A. de Kok W. G. Hol 《Protein science : a publication of the Protein Society》1997,6(4):913-915
Members of the family of 2-oxoacid dehydrogenase multienzyme complexes catalyze the oxidative decarboxylation of alpha-keto acids and are among the most remarkable enzymatic machineries in the living cell. These multienzyme complexes combine a highly symmetric (cubic or icosahedral) core with a dynamic and flexible arrangement of numerous subunits and domains surrounding the core. The center of the complex is formed by either 24 or 60 copies of dihydrolipoamide acetyltransferase (E2)-a multidomain enzyme. The hollow icosahedral cores are composed of 60 identical subunits of the catalytic domain of E2 with a molecular weight of about 1.8 million Da. Bipyramidal crystals suitable for X-ray diffraction of the icosahedral core of the pyruvate dehydrogenase multienzyme complex from Enterococcus faecalis were grown up to 0.7 mm in each dimension. The crystals belong to space group R32 with a = b = 244.3 A (hexagonal setting), and have a solvent content of 73%. The asymmetric unit contains one-third of the molecule, i.e., 20 of the 60 subunits. Initial X-ray crystallographic data to 7 A resolution were collected at cryotemperatures at synchrotron facilities. Interestingly, the diffraction was improved significantly upon rehydrating dehydrated crystals and extended to 4.2 A. 相似文献
9.
Shim da J Nemeria NS Balakrishnan A Patel H Song J Wang J Jordan F Farinas ET 《Biochemistry》2011,50(35):7705-7709
The first component (E1o) of the Escherichia coli 2-oxoglutarate dehydrogenase complex (OGDHc) was engineered to accept substrates lacking the 5-carboxylate group by subjecting H260 and H298 to saturation mutagenesis. Apparently, H260 is required for substrate recognition, but H298 could be replaced with hydrophobic residues of similar molecular volume. To interrogate whether the second component would allow synthesis of acyl-coenzyme A derivatives, hybrid complexes consisting of recombinant components of OGDHc (o) and pyruvate dehydrogenase (p) enzymes were constructed, suggesting that a different component is the "gatekeeper" for specificity for these two multienzyme complexes in bacteria, E1p for pyruvate but E2o for 2-oxoglutarate. 相似文献
10.
The thiamine-dependent E1o component (EC 1.2.4.2) of the 2-oxoglutarate dehydrogenase complex catalyses a rate-limiting step of the tricarboxylic acid cycle (TCA) of aerobically respiring organisms. We describe the crystal structure of Escherichia coli E1o in its apo and holo forms at 2.6 A and 3.5 A resolution, respectively. The structures reveal the characteristic fold that binds thiamine diphosphate and resemble closely the alpha(2)beta(2) hetero-tetrameric E1 components of other 2-oxo acid dehydrogenase complexes, except that in E1o, the alpha and beta subunits are fused as a single polypeptide. The extended segment that links the alpha-like and beta-like domains forms a pocket occupied by AMP, which is recognised specifically. Also distinctive to E1o are N-terminal extensions to the core fold, and which may mediate interactions with other components of the 2-oxoglutarate dehydrogenase multienzyme complex. The active site pocket contains a group of three histidine residues and one serine that appear to confer substrate specificity and the capacity to accommodate the TCA metabolite oxaloacetate. Oxaloacetate inhibits E1o activity at physiological concentrations, and we suggest that the inhibition may allow coordinated activity within the TCA cycle. We discuss the implications for metabolic control in facultative anaerobes, and for energy homeostasis of the mammalian brain. 相似文献
11.
The E. coli pyruvate and 2-oxoglutarate dehydrogenases are two closely related, large complexes that exemplify a growing number of multiprotein "machines" whose domains have been studied extensively and modeled in atomic detail, but whose quaternary structures have remained unclear for lack of an effective imaging technology. Here, electron cryotomography was used to show that the E1 and E3 subunits of these complexes are flexibly tethered approximately 11 nm away from the E2 core. This result demonstrates unambiguously that electron cryotomography can reveal the relative positions of features as small as 80 kDa in individual complexes, elucidating quaternary structure and conformational flexibility. 相似文献
12.
Pyruvate dehydrogenase (PDH), branched-chain 2-oxo acid dehydrogenase (BCDH) and 2-oxoglutarate dehydrogenase (OGDH) are multienzyme complexes that play crucial roles in several common metabolic pathways. These enzymes belong to a family of 2-oxo acid dehydrogenase complexes that contain multiple copies of three different components (E1, E2 and E3). For the Thermus thermophilus enzymes, depending on its substrate specificity (pyruvate, branched-chain 2-oxo acid or 2-oxoglutarate), each complex has distinctive E1 (E1p, E1b or E1o) and E2 (E2p, E2b or E2o) components and one of the two possible E3 components (E3b and E3o). (The suffixes, p, b and o identify their respective enzymes, PDH, BCDH and OGDH.) Our biochemical characterization demonstrates that only three specific E3*E2 complexes can form (E3b*E2p, E3b*E2b and E3o*E2o). X-ray analyses of complexes formed between the E3 components and the peripheral subunit-binding domains (PSBDs), derived from the corresponding E2-binding partners, reveal that E3b interacts with E2p and E2b in essentially the same manner as observed for Geobacillus stearothermophilus E3*E2p, whereas E3o interacts with E2o in a novel fashion. The buried intermolecular surfaces of the E3b*PSBDp/b and E3o*PSBDo complexes differ in size, shape and charge distribution and thus, these differences presumably confer the binding specificities for the complexes. 相似文献
13.
Rotational mobility of the lipoyl domain of a number of 2-oxoacid dehydrogenase complexes was investigated by transient dichroism after the domain had been specifically labeled with the triplet probe eosin-5-maleimide. Complexes investigated included pyruvate dehydrogenase complexes from Bacillus stearothermophilus, ox heart, and Escherichia coli (in which the E2 component had been genetically engineered to contain one lipoyl domain) and 2-oxoglutarate dehydrogenase complexes from ox heart and E. coli. Measurements were also performed with ox heart pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes specifically labeled on E1. Anisotropy decays were recorded in glycerol-buffer solutions of varying viscosity and at different temperatures. For E2-labeled complexes, the decays were found to be multiexponential, and the fastest correlation time was considerably shorter than expected for tumbling of the whole complex. This fast correlation time was absent from E1-labeled complexes and was assigned to independent motion of the lipoyl domain. Plots of the fast correlation time against eta/T showed a surprisingly weak dependence on viscosity and extrapolated to a time of 30-40 microseconds at zero viscosity. To explain this result, a model is proposed in which the lipoyl domain is in equilibrium between "free" and bound states. The time of 30-40 microseconds is shown to correspond to 1/koff, where koff is the rate constant for dissociation of the domain from binding sites on the complex. This dissociation phenomenon only contributes to the anisotropy decay when the viscosity of the solution is sufficiently high to slow the tumbling of the whole complex to times that are long in comparison to 1/koff. 相似文献
14.
G J Domingo H J Chauhan I A Lessard C Fuller R N Perham 《European journal of biochemistry》1999,266(3):1136-1146
The pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus was reconstituted in vitro from recombinant proteins derived from genes over-expressed in Escherichia coli. Titrations of the icosahedral (60-mer) dihydrolipoyl acetyltransferase (E2) core component with the pyruvate decarboxylase (E1, alpha2beta2) and dihydrolipoyl dehydrogenase (E3, alpha2) peripheral components indicated a variable composition defined predominantly by tight and mutually exclusive binding of E1 and E3 with the peripheral subunit-binding domain of each E2 chain. However, both analysis of the polypeptide chain ratios in complexes generated from various mixtures of E1 and E3, and displacement of E1 or E3 from E1-E2 or E3-E2 subcomplexes by E3 or E1, respectively, showed that the multienzyme complex does not behave as a simple competitive binding system. This implies the existence of secondary interactions between the E1 and E3 subunits and E2 that only become apparent on assembly. Exact geometrical distribution of E1 and E3 is unlikely and the results are best explained by preferential arrangements of E1 and E3 on the surface of the E2 core, superimposed on their mutually exclusive binding to the peripheral subunit-binding domain of the E2 chain. Correlation of the subunit composition with the overall catalytic activity of the enzyme complex confirmed the lack of any requirement for precise stoichiometry or strict geometric arrangement of the three catalytic sites and emphasized the crucial importance of the flexibility associated with the lipoyl domains and intramolecular acetyl group transfer in the mechanism of active-site coupling. 相似文献
15.
Sequences directing dihydrolipoamide dehydrogenase (E3) binding are located on the 2-oxoglutarate dehydrogenase (E1) component of the mammalian 2-oxoglutarate dehydrogenase multienzyme complex.
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Sequences located in the N-terminal region of the high M(r) 2-oxoglutarate dehydrogenase (E1) enzyme of the mammalian 2-oxoglutarate dehydrogenase multienzyme complex (OGDC) exhibit significant similarity with corresponding sequences from the lipoyl domains of the dihydrolipoamide acetyltransferase (E2) and protein X components of eukaryotic pyruvate dehydrogenase complexes (PDCs). Two additional features of this region of E1 resemble lipoyl domains: (i) it is readily released by trypsin, generating a small N-terminal peptide with an apparent M(r) value of 10,000 and a large stable 100,000 M(r) fragment (E1') and (ii) it is highly immunogenic, inducing the bulk of the antibody response to intact E1. This 'lipoyl-like' domain lacks a functional lipoamide group. Selective but extensive degradation of E1 with proteinase Arg C or specific conversion of E1 to E1' with trypsin both cause loss of overall OGDC function although the E1' fragment retains full catalytic activity. Removal of this small N-terminal peptide promotes the dissociation of dihydrolipoamide dehydrogenase (E3) from the E2 core assembly and also affects the stability of E1 interaction. Thus, structural roles which are mediated by a specific gene product, protein X in PDC and possibly also the E2 subunit, are performed by similar structural elements located on the E1 enzyme of the OGDC. 相似文献
16.
J K Stoops T S Baker J P Schroeter S J Kolodziej X D Niu L J Reed 《The Journal of biological chemistry》1992,267(34):24769-24775
Dihydrolipoamide acyltransferase (E2), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of alpha-keto acids, forms the central core to which the other components are attached. We have imaged by negative stain and cryoelectron microscopy the truncated dihydrolipoamide acetyltransferase core (60 subunits; M(r) = 2.7 x 10(6)) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex. Using icosahedral particle reconstruction techniques, we determined its structure to 25 A resolution. Although the model derived from the negative stain reconstruction was approximately 20% smaller than the model derived from the frozen-hydrated data, when corrected for the effects of the electron microscope contrast transfer functions, the reconstructions showed excellent correspondence. The pentagonal dodecahedron-shaped macromolecule has a maximum diameter, as measured along the 3-fold axis, of approximately 226 A (frozen-hydrated value), and 12 large openings (approximately 63 A in diameter) on the 5-fold axes that lead into a large solvent-accessible cavity (approximately 76-140 A diameter). The 20 vertices consist of cone-shaped trimers, each with a flattened base on the outside of the structure and an apex directed toward the center. The trimers are interconnected by 20 A thick "bridges" on the 2-fold axes. These studies also show that the highest resolution features apparent in the frozen-hydrated reconstruction are revealed in a filtered reconstruction of the stained molecule. 相似文献
17.
Induction of the branched-chain 2-oxo acid dehydrogenase complex in 3T3-L1 adipocytes during differentiation. 总被引:5,自引:1,他引:4
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The activities of 2-oxo acid dehydrogenase complexes were measured during hormone-mediated differentiation of 3T3-L1 preadipocytes into adipocytes. Specific activity of leucine-activated branched-chain 2-oxo acid dehydrogenase complex increased approx. 10-fold in 3T3-L1 adipocytes compared with 3T3-L1 preadipocytes. In contrast, specific activity of the 2-oxoglutarate dehydrogenase complex increased by only 3-fold in 3T3-L1 adipocytes. The three catalytic component enzymes of the branched-chain 2-oxo acid dehydrogenase complex and the pyruvate dehydrogenase complex showed concomitant increases in their specific activities. A close similarity in kinetics of induction of the branched-chain 2-oxo acid dehydrogenase complex and the pyruvate dehydrogenase complex in 3T3-L1 adipocytes suggests that a common mechanism may be involved in hormone-dependent increases in the activities of the catalytic components of these two complexes in 3T3-L1 adipocytes during differentiation. 相似文献
18.
The subunits of the dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex can form a 60-mer via association of the C-terminal I domain of E2 at the vertices of a dodecahedron. Exterior to this inner core structure, E2 has a pyruvate dehydrogenase component (E1)-binding domain followed by two lipoyl domains, all connected by mobile linker regions. The assembled core structure of mammalian pyruvate dehydrogenase complex also includes the dihydrolipoyl dehydrogenase (E3)-binding protein (E3BP) that binds the I domain of E2 by its C-terminal I' domain. E3BP similarly has linker regions connecting an E3-binding domain and a lipoyl domain. The composition of E2.E3BP was thought to be 60 E2 plus approximately 12 E3BP. We have prepared homogenous human components. E2 and E2.E3BP have s(20,w) values of 36 S and 31.8 S, respectively. Equilibrium sedimentation and small angle x-ray scattering studies indicate that E2.E3BP has lower total mass than E2, and small angle x-ray scattering showed that E3 binds to E2.E3BP outside the central dodecahedron. In the presence of saturating levels of E1, E2 bound approximately 60 E1 and maximally sedimented 64.4 +/- 1.5 S faster than E2, whereas E1-saturated E2.E3BP maximally sedimented 49.5 +/- 1.4 S faster than E2.E3BP. Based on the impact on sedimentation rates by bound E1, we estimate fewer E1 (approximately 12) were bound by E2.E3BP than by E2. The findings of a smaller E2.E3BP mass and a lower capacity to bind E1 support the smaller E3BP substituting for E2 subunits rather than adding to the 60-mer. We describe a substitution model in which 12 I' domains of E3BP replace 12 I domains of E2 by forming 6 dimer edges that are symmetrically located in the dodecahedron structure. Twelve E3 dimers were bound per E248.E3BP12 mass, which is consistent with this model. 相似文献
19.
The state of assembly of the pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes was examined after the dihydrolipoyl acyltransferase (E2) component of each enzyme system had been subjected to varying degrees of limited proteolysis. Dissociation of the dihydrolipoyl dehydrogenase (E3) component accompanied specifically the excision of a homologous segment of each E2 chain that connects the N-terminal lipoyl domain(s) with a C-terminal catalytic domain. The latter remains aggregated as a 24-mer and retains its capacity to bind the 2-oxo-acid decarboxylase (E1) component. The relevant segment of the E2o chain from the 2-oxoglutarate dehydrogenase complex was isolated and shown to be a folded protein which still binds to E3. 相似文献
20.
Purification of 2-oxo acid dehydrogenase multienzyme complexes from ox heart by a new method. 总被引:15,自引:5,他引:10
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A new method is described that allows the parallel purification of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart without the need for prior isolation of mitochondria. All the assayable activity of the 2-oxo acid dehydrogenase complexes in the disrupted tissue is made soluble by the inclusion of non-ionic detergents such as Triton X-100 or Tween-80 in the buffer used for the initial extraction of the enzyme complexes. The yields of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes are many times greater than those obtained by means of previous methods. In terms of specific catalytic activity, banding pattern on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, sedimentation properties and possession of the regulatory phosphokinase bound to the pyruvate dehydrogenase complex, the 2-oxo acid dehydrogenase complexes prepared by the new method closely resemble those described by previous workers. The greatly improved yield of 2-oxo acid dehydrogenase complexes occasioned by the use of Triton X-100 or Tween-80 as solubilizing agent supports the possibility that the bulk of the pyruvate dehydrogenase complex is associated in some way with the mitochondrial inner membrane and is not free in the mitochondrial matrix space. 相似文献