Configuration of interdomain linkers in pyruvate dehydrogenase complex of Escherichia coli as determined by cryoelectron microscopy.

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.     

Component X of mammalian pyruvate dehydrogenase complex: structural and functional relationship to the lipoate acetyltransferase (E2) component

The lipoate acetyltransferase (E2, Mr 70,000) and protein X (Mr 51,000) subunits of the bovine pyruvate dehydrogenase multienzyme complex (PDC) core assembly are antigenically distinct polypeptides. However comparison of the N-terminal amino acid sequence of the E2 and X polypeptides reveals significant homology between the two components. Selective tryptic release of the 14C-labelled acetylated lipoyl domains of E2 and protein X from native PDC generates stable, radiolabelled 34 and 15 kDa fragments, respectively. Thus, in contrast to E2 which contains two tandemly-arranged lipoyl domains, protein X appears to contain only a single lipoyl domain located at its N-terminus.     

Isolation and characterisation of the pyruvate dehydrogenase complex of anaerobically grown Enterococcus faecalis NCTC 775.

In this contribution the isolation and some of the structural and kinetic properties of the pyruvate dehydrogenase complex (PDC) of anaerobically grown Enterococcus faecalis are described. The complex closely resembles the PDC of other Gram-positive bacteria and eukaryotes. It consists of four polypeptide chains with apparent molecular masses on SDS/PAGE of 97, 55, 42 and 36 kDa, and these polypeptides could be assigned to dihydrolipoyl transacetylase (E2), lipoamide dehydrogenase (E3) and the two subunits of pyruvate dehydrogenase (E1 alpha and E1 beta), respectively. The E2 core has an icosahedral symmetry. The apparent molecular mass on SDS/PAGE of 97 kDa of the E2 chain is extremely high in comparison with other Gram-positive organisms (and eukaryotes) and probably due to several lipoyl domains associated with the E2 chain. NADH inhibition is mediated via E3. The mechanism of inhibition is discussed in view of the high PDC activities in vivo that are found in E. faecalis, grown under anaerobic conditions.     

Subunit and Catalytic Component Stoichiometries of an in Vitro Reconstituted Human Pyruvate Dehydrogenase Complex

The human pyruvate dehydrogenase complex (PDC) is a 9.5-megadalton catalytic machine that employs three catalytic components, i.e. pyruvate dehydrogenase (E1p), dihydrolipoyl transacetylase (E2p), and dihydrolipoamide dehydrogenase (E3), to carry out the oxidative decarboxylation of pyruvate. The human PDC is organized around a 60-meric dodecahedral core comprising the C-terminal domains of E2p and a noncatalytic component, E3-binding protein (E3BP), which specifically tethers E3 dimers to the PDC. A central issue concerning the PDC structure is the subunit stoichiometry of the E2p/E3BP core; recent studies have suggested that the core is composed of 48 copies of E2p and 12 copies of E3BP. Here, using an in vitro reconstituted PDC, we provide densitometry, isothermal titration calorimetry, and analytical ultracentrifugation evidence that there are 40 copies of E2p and 20 copies of E3BP in the E2p/E3BP core. Reconstitution with saturating concentrations of E1p and E3 demonstrated 40 copies of E1p heterotetramers and 20 copies of E3 dimers associated with the E2p/E3BP core. To corroborate the 40/20 model of this core, the stoichiometries of E3 and E1p binding to their respective binding domains were reexamined. In these binding studies, the stoichiometries were found to be 1:1, supporting the 40/20 model of the core. The overall maximal stoichiometry of this in vitro assembled PDC for E2p:E3BP:E1p:E3 is 40:20:40:20. These findings contrast a previous report that implicated that two E3-binding domains of E3BP bind simultaneously to a single E3 dimer (Smolle, M., Prior, A. E., Brown, A. E., Cooper, A., Byron, O., and Lindsay, J. G. (2006) J. Biol. Chem. 281, 19772–19780).The human pyruvate dehydrogenase complex (PDC)³ resides in mitochondria and catalyzes the oxidative decarboxylation of pyruvate to yield acetyl-CoA and reducing equivalents (NADH), serving as a link between glycolysis and the Krebs cycle (1–3). The PDC is a large (∼9.5 MDa) catalytic machine comprising multiple protein components. The three catalytic components are pyruvate dehydrogenase (E1p), dihydrolipoyl transacetylase (E2p), and dihydrolipoamide dehydrogenase (E3), with E3 being a common component between different α-keto acid dehydrogenase complexes. The two regulatory enzymes in the PDC are the isoforms of pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.The PDC is organized around a structural core, which includes the C-terminal domains of E2p and a noncatalytic component that specifically binds E3, i.e. the E3-binding protein (E3BP). To this E2p/E3BP core, multiple copies of the other PDC components are tethered through noncovalent interactions. Each E2p subunit contains two consecutive N-terminal lipoic acid-bearing domains (LBDs), termed L1 and L2, followed by the E1p-binding domain (E1pBD) and the C-terminal inner-core/catalytic domain, with these independent domains connected by unstructured linkers. Similarly, each E3BP subunit consists of a single N-terminal LBD (referred to as L3), the E3-binding domain (E3BD), and the noncatalytic inner core domain. Together, the inner core domains of E2p and E3BP assemble to form the dodecahedral 60-meric E2p/E3BP core. The role of the E1pBD and E3BD domains is to tether E1p and E3, respectively, to the periphery of the E2p/E3BP core. It is presumed that the LBDs (L1, L2, and L3) shuttle between the active sites of the three catalytic components of the PDC during the oxidative decarboxylation cycle (4). The eukaryotic PDC is unique among α-keto acid dehydrogenase complexes in its requirement for E3BP; prokaryotic PDCs employ the single subunit-binding domain to secure either E1p or E3 to the complex (5).Using a “divide-and-conquer” approach, a wealth of structural information on the PDC has been accumulated recently. High-resolution crystal structures are available for the human E1p (6–8) and E3 components (9). A model for the human E2p has been constructed based on an 8.8-Å electron density map available from cryo-electron microscopy (10). Additionally, solution and crystal structures of the L1 and L2 domains of E2p have been determined (11–13), and the high-resolution crystal structures of the E3BD (14, 15), pyruvate dehydrogenase kinase isoforms 1–4 (12, 16–18), and pyruvate dehydrogenase phosphatase isoform 1 (19) are known. Therefore, atomic models are available for almost all components and domains of the mammalian PDC.With the successes of the above structural approach, attention has turned to the overall structure of the PDC. There are two outstanding questions as follows. What are the subunit and overall catalytic component stoichiometries? What are the positions and orientations of the components in this large catalytic machine? Yu et al. (10) recently determined the cryo-EM structure of a PDC core comprising only human E2p subunits. Like yeast E2p, human E2p adopts a dodecahedral structure composed of 60 E2p proteins; each face of the dodecahedron has a large gap. Although this structure is highly informative, the composition of this core deviates substantially from that of the native PDC, because no E3BP subunits are present in the core structure. Based on the similar structure of the dodecahedral yeast PDC, a hypothesis was formed that, in human PDC, 12 copies of E3BP bind in the 12 gaps, which is termed the “60/12” model (20). Biophysical studies on complexes of E2p and E3BP later negated the 60/12 model; Hiromasa et al. (21) therefore posited an alternative, the “48/12” model, in which the dodecahedral core includes 48 E2p subunits and 12 E3BP proteins. A further source of conjecture is how many E1p and E3 components bind to the periphery of the PDC. If one binding domain binds to one peripheral catalytic component, a maximally occupied 60/12 PDC would harbor 60 E1p heterotetramers and 12 E3 dimers (or 48 E1ps and 12 E3s in the 48/12 model). The notion of such 1:1 binding is supported by the preponderance of available biophysical evidence. Specifically, two crystal structures, site-directed mutagenesis, and calorimetric measurements describe a 1:1 interaction between E3BD and E3 (14, 15). Also, although no structures are available for the human E1p-E1pBD complex, a crystal structure of the homologs of these proteins from Bacillus stearothermophilus also demonstrates a 1:1 interaction between the E1pBD of E2p and the E1p heterotetramer (22). In addition, ITC experiments performed on the bacterial E1p and the cognate subunit-binding domain indicate a 1:1 association (23). At variance with the above observations, a different subunit stoichiometry has been proposed by Smolle et al. (24, 25). Their evidence suggests that two binding domains bind for every peripheral component; such an arrangement potentially yields a PDC with half as many peripheral components bound.This study was undertaken to ascertain the subunit and component stoichiometries of the human PDC, particularly with regard to interactions between the E3BD and the E3 dimer. We show that quantification of bands on an SDS-polyacrylamide gel of a PDC reconstituted at saturating E1p and E3 concentrations supports neither the 60/12 nor the 48/12 model. Instead, a “40/20” model is proposed, and subsequent ITC and analytical ultracentrifugation (AUC) data corroborate this new model. In addition, results from electrophoretic mobility shift assays, ITC, and AUC presented here uniformly show a 1:1 interaction between E3BD and the E3 dimer as well as between E1pBD and the E1p heterotetramer. The implications of this 1:1 binding stoichiometry for the macromolecular assembly of the PDC are discussed.     

A new level of architectural complexity in the human pyruvate dehydrogenase complex

Mammalian pyruvate dehydrogenase multienzyme complex (PDC) is a key metabolic assembly comprising a 60-meric pentagonal dodecahedral E2 (dihydrolipoamide acetyltransferase) core attached to which are 30 pyruvate decarboxylase E1 heterotetramers and 6 dihydrolipoamide dehydrogenase E3 homodimers at maximal occupancy. Stable E3 integration is mediated by an accessory E3-binding protein (E3BP) located on each of the 12 E2 icosahedral faces. Here, we present evidence for a novel subunit organization in which E3 and E3BP form subcomplexes with a 1:2 stoichiometry implying the existence of a network of E3 "cross-bridges" linking pairs of E3BPs across the surface of the E2 core assembly. We have also determined a low resolution structure for a truncated E3BP/E3 subcomplex using small angle x-ray scattering showing one of the E3BP lipoyl domains docked into the E3 active site. This new level of architectural complexity in mammalian PDC contrasts with the recently published crystal structure of human E3 complexed with its cognate subunit binding domain and provides important new insights into subunit organization, its catalytic mechanism and regulation by the intrinsic PDC kinase.     

Cryoelectron microscopy of mammalian pyruvate dehydrogenase complex.

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.     

Crystal structure of pyruvate dehydrogenase kinase 3 bound to lipoyl domain 2 of human pyruvate dehydrogenase complex

The human pyruvate dehydrogenase complex (PDC) is regulated by reversible phosphorylation by four isoforms of pyruvate dehydrogenase kinase (PDK). PDKs phosphorylate serine residues in the dehydrogenase (E1p) component of PDC, but their amino-acid sequences are unrelated to eukaryotic Ser/Thr/Tyr protein kinases. PDK3 binds to the inner lipoyl domains (L2) from the 60-meric transacetylase (E2p) core of PDC, with concomitant stimulated kinase activity. Here, we present crystal structures of the PDK3-L2 complex with and without bound ADP or ATP. These structures disclose that the C-terminal tail from one subunit of PDK3 dimer constitutes an integral part of the lipoyl-binding pocket in the N-terminal domain of the opposing subunit. The two swapped C-terminal tails promote conformational changes in active-site clefts of both PDK3 subunits, resulting in largely disordered ATP lids in the ADP-bound form. Our structural and biochemical data suggest that L2 binding stimulates PDK3 activity by disrupting the ATP lid, which otherwise traps ADP, to remove product inhibition exerted by this nucleotide. We hypothesize that this allosteric mechanism accounts, in part, for E2p-augmented PDK3 activity.     

The pyruvate dehydrogenase complex from the parasitic nematode Ascaris suum: novel subunit composition and domain structure of the dihydrolipoyl transacetylase component.

The pyruvate dehydrogenase complex (PDC) from muscle of the adult parasitic nematode Ascaris suum plays a unique role in its anaerobic mitochondrial metabolism. Resolution of the intact complex in high salt dissociates the pyruvate dehydrogenase subunit but leaves the dihydrolipoyl dehydrogenase subunit (E3) and two other proteins with apparent M(r)s of 45 and 43 kDa bound to the dihydrolipoyl transacetylase (E2) core. These proteins are not observable on Coomassie brilliant blue-stained gels of other eukaryotic PDCs, but the 45-kDa protein is similar in apparent M(r), pI, and sensitivity to trypsin to the Kb subunit of the bovine kidney PDH alpha kinase. Acetylation of the ascarid PDC with [2-14C]pyruvate under conditions designed to maximize the incorporation of label into protein yielded only a single radiolabeled subunit, E2. These results confirm earlier reports that the ascarid PDC lacks protein X, an integral component recently identified in other eukaryotic PDCs. About 1.6 to 1.8 mol of 14C was incorporated/mole of E2, suggesting that the ascarid E2 contained two lipoly-bearing domains. Domain mapping of the 14C-acetylated ascarid E2 by limited tryptic digestion identified two lipoyl-bearing fragments with apparent M(r)s of 50 and 34 kDa and two core fragments with apparent M(r)s of 46 and 30 kDa. The ascarid E2 domain structure appears to be similar to that of other E2s. However, it appears that the subunit-binding domain (E2B) of the ascarid E2 may be significantly larger or be flanked by larger than normal interdomain regions. An enlarged E2B domain may be necessary to accommodate the additional binding of E3 to the E2 subunit in the ascarid complex, in the absence of protein X.     

Immunogenetic analysis reveals that epitope shifting occurs during B-cell affinity maturation in primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is a liver disease characterized by serum autoantibodies against the pyruvate dehydrogenase complex (PDC) located in the inner mitochondrial membrane. The predominant target in PDC has previously been localized to the inner lipoyl domain (ILD) of the E2 subunit. The etiology of PBC is unknown, although molecular mimicry with bacterial PDC has been proposed. Here, we have investigated the etiology of PBC and nature of the autoimmune response by analyzing the structure of a human monoclonal antibody with ILD specificity. Mutants of the monoclonal antibody, which was originally isolated from a patient with PBC, were expressed as Fab by phage display, and tested for reactivity against recombinant domains of the E2 subunit. Fab in which the V(H)-encoded portions were reverted to germline lost reactivity against the ILD alone, but recognized a different epitope in a didomain construct encompassing the ILD, hinge region and E1/E3 binding domain. The complete V(H) and V(L )germline revertant was unreactive with the human ILD and didomain, the Escherichia coli didomain, and whole PDC. We hypothesize that the IgM on the surface of the na?ve B-cell first recognizes an as yet unidentified antigen, and that accumulation of somatic mutations results in an intermolecular epitope shift directed towards an epitope involving the E1/E3 binding domain. Further mutations result in the specificity being redirected to the ILD. These findings also suggest that bacterial molecular mimicry is not involved in initiating disease.     

The dihydrolipoamide S-acetyltransferase subunit of the mitochondrial pyruvate dehydrogenase complex from maize contains a single lipoyl domain.

The dihydrolipoamide S-acetyltransferase (E2) subunit of the maize mitochondrial pyruvate dehydrogenase complex (PDC) was postulated to contain a single lipoyl domain based upon molecular mass and N-terminal protein sequence (Thelen, J. J., Miernyk, J. A., and Randall, D. D. (1998) Plant Physiol. 116, 1443-1450). This sequence was used to identify a cDNA from a maize expressed sequence tag data base. The deduced amino acid sequence of the full-length cDNA was greater than 30% identical to other E2s and contained a single lipoyl domain. Mature maize E2 was expressed in Escherichia coli and purified to a specific activity of 191 units mg(-1). The purified recombinant protein had a native mass of approximately 2.7 MDa and assembled into a 29-nm pentagonal dodecahedron as visualized by electron microscopy. Immunoanalysis of mitochondrial proteins from various plants, using a monoclonal antibody against the maize E2, revealed 50-54-kDa cross-reacting polypeptides in all samples. A larger protein (76 kDa) was also recognized in an enriched pea mitochondrial PDC preparation, indicating two distinct E2s. The presence of a single lipoyl-domain E2 in Arabidopsis thaliana was confirmed by identifying a gene encoding a hypothetical protein with 62% amino acid identity to the maize homologue. These data suggest that all plant mitochondrial PDCs contain an E2 with a single lipoyl domain. Additionally, A. thaliana and other dicots possess a second E2, which contains two lipoyl domains and is only 33% identical at the amino acid level to the smaller isoform. The reason two distinct E2s exist in dicotyledon plants is uncertain, although the variability between these isoforms, particularly within the subunit-binding domain, suggests different roles in assembly and/or function of the plant mitochondrial PDC.     

Salt-Induced Changes in the Subunit Structure of the Bacillus stearothermophilus Lipoate Acetyltransferase

The Bacillus stearothermophilus lipoate acetyltransferase (E2), composed of sixty identical, subunits is the core component of the pyruvate dehydrogenase complex (PDC). E2 polypeptide is composed of LD, PSBD, and CD domains. Most studies had focused on a truncated E2 that is deficient in LD and PSBD, because CD mainly contributes to maintaining the multimeric structure. We examined salt-induced changes in E2 without truncation and constructed reaction models. We speculate that in the presence of KCl, E2 is dissociated into a monomer and then assembled into an aggregative complex (C_A) and a quasi-stable complex (C_Q). C_A was larger than C_Q, but smaller than intact E2. C_A and C_Q were dominant complexes at about neutral pH and at basic pH respectively. PDC, in which PSBD is occupied by other components, and a truncated E2 undergo dissociation only. LD-PSBD region besides CD might then contribute to the partial association of dissociated E2.     

Binding of pyruvate dehydrogenase to the core of the human pyruvate dehydrogenase complex

In human (h) pyruvate dehydrogenase complex (PDC) the pyruvate dehydrogenase (E1) is bound to the E1-binding domain of dihydrolipoamide acetyltransferase (E2). The C-terminal surface of the E1beta subunit was scanned for the negatively charged residues involved in binding with E2. betaD289 of hE1 interacts with K276 of hE2 in a manner similar to the corresponding interaction in Bacillus stearothermophilus PDC. In contrast to bacterial E1beta, the C-terminal residue of the hE1beta does not participate in the binding with positively charged residues of hE2. This latter finding shows species specificity in the interaction between hE1beta and hE2 in PDC.     

Recognition of the inner lipoyl-bearing domain of dihydrolipoyl transacetylase and of the blood glucose-lowering compound AZD7545 by pyruvate dehydrogenase kinase 2

Pyruvate dehydrogenase kinase 2 (PDHK2) is a unique mitochondrial protein kinase that regulates the activity of the pyruvate dehydrogenase multienzyme complex (PDC). PDHK2 is an integral component of PDC tightly bound to the inner lipoyl-bearing domains (L2) of the dihydrolipoyl transacetylase component (E2) of PDC. This association has been reported to bring about an up to 10-fold increase in kinase activity. Despite the central role played by E2 in the maintenance of PDHK2 functionality in the PDC-bound state, the molecular mechanisms responsible for the recognition of L2 by PDHK2 and for the E2-dependent PDHK2 activation are largely unknown. In this study, we used a combination of molecular modeling and site-directed mutagenesis to identify the amino acid residues essential for the interaction between PDHK2 and L2 and for the activation of PDHK2 by E2. On the basis of the results of site-directed mutagenesis, it appears that a number of PDHK2 residues located in its R domain (P22, L23, F28, F31, F44, L45, and L160) and in the so-called "cross arm" structure (K368, R372, and K391) are critical in determining the strength of the interaction between PDHK2 and L2. The residues of L2 essential for recognition by PDHK2 include L140, K173, I176, E179, and to a lesser extent D164, D172, and A174. Importantly, certain PDHK2 residues forming interfaces with L2, i.e., K17, P22, F31, F44, R372, and K391, are also critical for the maintenance of enhanced PDHK2 activity in the E2-bound state. Finally, evidence that the blood glucose-lowering compound AZD7545 disrupts the interactions between PDHK2 and L2 and thereby inhibits PDHK2 activity is presented.     

Protection by thiols of the mitochondrial complexes from 4-hydroxy-2-nonenal

In the present study, the effects of 4-hydroxy-2-nonenal (HNE) on highly purified pyruvate dehydrogenase complex (PDC) and its catalytic components in vitro and on PDC, alpha-ketoglutarate dehydrogenase complex (KGDC), and the branched-chain alpha-keto acid dehydrogenase complex (BCKDC) activities in cultured human HepG2 cells were investigated. Among the PDC components, the activity of the dihydrolipoamide acetyltransferase-E3-binding protein subcomplex (E2-E3BP) only was decreased by HNE. Dihydrolipoamide dehydrogenase (E3) protected the E2-E3BP subcomplex from HNE inactivation in the absence of the substrates. In the presence of E3 and NADH, when lipoyl groups were reduced, higher inactivation of the E2-E3BP subcomplex by HNE was observed. Purified PDC was protected from HNE-induced inactivation by several thiol compounds including lipoic acid plus [LA-plus; 2-(N,N-dimethylamine)ethylamidolipoate(.)HCl]. Treatment of cultured HepG2 cells with HNE resulted in a significant reduction of PDC and KGDC activities, whereas BCKDC activity decreased to a lesser extent. Lipoyl compounds afforded protection from HNE-induced inhibition of PDC. This protection was higher in the presence of cysteine and reduced glutathione. Cysteine was able to restore PDC activity to some extent after HNE treatment. These findings show that thiols, including lipoic acid, provide protection against HNE-induced inactivation of lipoyl-containing complexes in the mitochondria.     

Purification and cellular localization of wild type and mutated dihydrolipoyltransacetylases from Azotobacter vinelandii and Escherichia coli expressed in E. coli.

Wild type dihydrolipoyltransacetylase(E2p)-components from the pyruvate dehydrogenase complex of A. vinelandii or E. coli, and mutants of A. vinelandii E2p with stepwise deletions of the lipoyl domains or the alanine- and proline-rich region between the binding and the catalytic domain have been overexpressed in E. coli TG2. The high expression of A. vinelandii wild type E2p (20% of cellular protein) and of a mutant enzyme with two lipoyl domains changed the properties of the inner bacterial membrane. This resulted in a solubilization of A. vinelandii E2p after degradation of the outer membrane by lysozyme without any contamination by E. coli pyruvate dehydrogenase complex (PDC) or other high-molecular-weight contaminants. The same effect could be detected for A. vinelandii E2o, an E2 which contains only one lipoyl domain, whereas almost no solubilization of A. vinelandii E2p with one lipoyl domain or of E2p consisting only of the binding and catalytic domain was found. Partial or complete deletion of the alanine- and proline-rich sequence between the binding and the catalytic domain did also decrease the solubilization of the E2p-mutants after lysozyme treatment. Immunocytochemical experiments on E. coli TG2 cells expressing A. vinelandii wild type E2p indicated that the enzyme was present as a soluble protein in the cytoplasm. In contrast, overexpressed A. vinelandii E2p with deletion of all three lipoyl domains and E. coli wild type E2p aggregated intracellularly. The solubilization by lysozyme is therefore ascribed to excluded volume effects leading to changes in the properties of the inner bacterial membrane.     

Sequences directing dihydrolipoamide dehydrogenase (E3) binding are located on the 2-oxoglutarate dehydrogenase (E1) component of the mammalian 2-oxoglutarate dehydrogenase multienzyme complex.

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.     

R-Lipoic Acid Inhibits Mammalian Pyruvate Dehydrogenase Kinase

The four pyruvate dehydrogenase kinase (PDK) and two pyruvate dehydrogenase phosphatase (PDP) isoenzymes that are present in mammalian tissues regulate activity of the pyruvate dehydrogenase complex (PDC) by phosphorylation/dephosphorylation of its pyruvate dehydrogenase (E1) component. The effect of lipoic acids on the activity of PDKs and PDPs was investigated in purified proteins system. R-lipoic acid, S-lipoic acid and R-dihydrolipoic acid did not significantly affect activities of PDPs and at the same time inhibited PDKs to different extents (PDK1?>?PDK4?～?PDK2?>?PDK3 for R-LA). Since lipoic acids inhibited PDKs activity both when reconstituted in PDC and in the presence of E1 alone, dissociation of PDK from the lipoyl domains of dihydrolipoamide acetyltransferase in the presence of lipoic acids is not a likely explanation for inhibition. The activity of PDK1 towards phosphorylation sites 1, 2 and 3 of E1 was decreased to the same extent in the presence of R-lipoic acid, thus excluding protection of the E1 active site by lipoic acid from phosphorylation. R-lipoic acid inhibited autophosphorylation of PDK2 indicating that it exerted its effect on PDKs directly. Inhibition of PDK1 by R-lipoic acid was not altered by ADP but was decreased in the presence of pyruvate which itself inhibits PDKs. An inhibitory effect of lipoic acid on PDKs would result in less phosphorylation of E1 and hence increased PDC activity. This finding provides a possible mechanism for a glucose (and lactate) lowering effect of R-lipoic acid in diabetic subjects.     

R-lipoic acid inhibits mammalian pyruvate dehydrogenase kinase

The four pyruvate dehydrogenase kinase (PDK) and two pyruvate dehydrogenase phosphatase (PDP) isoenzymes that are present in mammalian tissues regulate activity of the pyruvate dehydrogenase complex (PDC) by phosphorylation/dephosphorylation of its pyruvate dehydrogenase (E1) component. The effect of lipoic acids on the activity of PDKs and PDPs was investigated in purified proteins system. R-lipoic acid, S-lipoic acid and R-dihydrolipoic acid did not significantly affect activities of PDPs and at the same time inhibited PDKs to different extents (PDK1>PDK4 approximately PDK2>PDK3 for R-LA). Since lipoic acids inhibited PDKs activity both when reconstituted in PDC and in the presence of E1 alone, dissociation of PDK from the lipoyl domains of dihydrolipoamide acetyltransferase in the presence of lipoic acids is not a likely explanation for inhibition. The activity of PDK1 towards phosphorylation sites 1, 2 and 3 of E1 was decreased to the same extent in the presence of R-lipoic acid, thus excluding protection of the E1 active site by lipoic acid from phosphorylation. R-lipoic acid inhibited autophosphorylation of PDK2 indicating that it exerted its effect on PDKs directly. Inhibition of PDK1 by R-lipoic acid was not altered by ADP but was decreased in the presence of pyruvate which itself inhibits PDKs. An inhibitory effect of lipoic acid on PDKs would result in less phosphorylation of E1 and hence increased PDC activity. This finding provides a possible mechanism for a glucose (and lactate) lowering effect of R-lipoic acid in diabetic subjects.     

Regulation of pyruvate dehydrogenase complex activity in Ehrlich ascites carcinoma cells by Ca2+ and pyruvate

The dependence of pyruvate dehydrogenase complex (PDC) activity on [Ca2+] was determined in Ehrlich ascites carcinoma cells at different pyruvate concentrations. The resulting family of curves had the following characteristics: a) bell-shaped appearance of all curves with maximum activity at 600 nM Ca2+; b) unchanged position of maxima with changes in pyruvate concentration; c) nonmonotonous changes in PDC activity with increasing pyruvate concentration at fixed [Ca2+]. Feasible mechanisms involving Ca2+-dependent phosphatase and kinase which are consistent with the experimental findings are discussed. To determine the steps in the chain of PDC reactions which determine the observed phenomena, a mathematical model is suggested which is based on the known data on the structural--functional relationships between the complex components--pyruvate dehydrogenase (E1), dihydrolipoyl acetyl transferase (E2), dihydrolipoyl dehydrogenase (E3), protein X, kinase, and phosphatase. To adequately describe the non-trivial dependence of PDC activity on [Ca2+] at different pyruvate concentrations, it was also necessary to consider the interdependence of some steps in the general chain of PDC reactions. Phenomenon (a) is shown to be due only to the involvement of protein X in the PDC reactions, phenomenon (b) to be due to changes in the activity of kinase, and phenomenon (c) to be due to dependence of acetylation and transacetylation rates on pyruvate concentration.