A computer model analysis of the active-site coupling mechanism in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

A computer modeling system developed to analyze experimental data for inactivation of the Escherichia coli alpha-ketoglutarate dehydrogenase complex (KGDC) accompanying release of lipoyl moieties by lipoamidase and by trypsin [Hackert, M.L., Oliver, R.M. & Reed, L.J. (1983) Proc. Natl. Acad. Sci. USA 80, 2226-2230] was used to analyze analogous data for the E. coli pyruvate dehydrogenase complex (PDC). The model studies indicate that the activity of PDC, as found for KGDC, is influenced by redundancies and random processes, which we describe as a multiple random coupling mechanism. In both complexes more than one lipoyl moiety services each pyruvate dehydrogenase (EC 1.2.4.1) or alpha-ketoglutarate dehydrogenase (EC 1.2.4.2) (E1) subunit, and an extensive lipoyl-lipoyl interaction network for exchange of electrons and possibly acyl groups must also be present. The best fit between computed and experimental data for PDC was obtained with a model that has four lipoyl domains with four or, more probably, eight lipoyl moieties servicing each E1 subunit. The lipoyl-lipoyl interaction network for PDC has lipoyl domain interactions similar to those found for KGDC plus the additional possibility of interaction of a lipoyl moiety and its paired mate on each dihydrolipoamide acetyltransferase (EC 2.3.1.12) (E2) subunit. The two lipoyl moieties on an E2 subunit in PDC appear to be functionally indistinguishable, each servicing the acetyltransferase site of that E2 subunit and a dihydrolipoamide dehydrogenase (EC 1.6.4.3) (E3) subunit if the latter is bound to that particular E2 subunit. The observed difference between inactivation of PDC by lipoamidase and by trypsin appears to be due to dead-end competitive inhibition by lipoyl domains that have been modified by excision of lipoyl moieties by lipoamidase.     

Acyl group and electron pair relay system: A network of interacting lipoyl moieties in the pyruvate and α-ketoglutarate dehydrogenase complexes from Escherichia coli

The dihydrolipoyl transacetylase component of the Escherichia coli pyruvate dehydrogenase complex [pyruvate:lipoate oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1] bears two sites on each of its 24 polypeptide chains that undergo reductive acetylation by [2-(14)C]pyruvate and thiamin pyrophosphate, acetylation by [1-(14)C]acetyl-CoA in the presence of DPNH, and reaction with N-ethyl[2,3-(14)C]maleimide in the presence of pyruvate and thiamin pyrophosphate. The data strongly imply that these sites are covalently bound lipoyl moieties. The results of similar experiments with the E. coli alpha-ketoglutarate dehydrogenase complex [2-oxoglutarate:lipoate oxidoreductase (decarboxylating and acceptor-succinylating), EC 1.2.4.2] indicate that its dihydrolipoyl transsuccinylase component bears only one lipoyl moiety on each of its 24 chains. Charging of the 48 acetyl acceptor sites on the transacetylase or the 24 succinyl acceptor sites on the transsuccinylase by pyruvate or alpha-ketoglutarate, respectively, and thiamin pyrophosphate was observed in the presence of only a few functionally active pyruvate dehydrogenase or alpha-ketoglutarate dehydrogenase chains. Extensive crosslinking of the transacetylase chains was observed when the pyruvate dehydrogenase complex was treated with pyruvate and thiamin pyrophosphate or with DPNH in the presence of N,N'-o- or N,N'-p-phenylenedimaleimide, respectively. When the alpha-ketoglutarate dehydrogenase complex was treated with DPNH in the presence of N,N'-p-phenylenedimaleimide, only transsuccinylase monomers and crosslinked transsuccinylase dimers were detected. It appears that the 48 lipoyl moieties in the transacetylase and the 24 lipoyl moieties in the transsuccinylase comprise an interacting network that functions as an acyl group and electron pair relay system through thiol-disulfide and acyl-transfer reactions among all of the lipoyl moieties.     

Evidence for a multiple random coupling mechanism in the alpha-ketoglutarate dehydrogenase multienzyme complex of Escherichia coli: a computer model analysis.

A computer modeling system was used to analyze experimental data for inactivation of the Escherichia coli alpha-ketoglutarate dehydrogenase complex accompanying release of lipoic acid residues by lipoamidase and by trypsin [Stepp, L. R., Bleile, D. M., McRorie, D. K., Pettit, F. H. & Reed, L. J. (1981) Biochemistry 20, 4555-4560]. The results provide insight into the active-site coupling mechanism in the alpha-ketoglutarate dehydrogenase complex. The model studies indicate that the overall activity of the alpha-ketoglutarate dehydrogenase complex is influenced by redundancies and random processes that we describe as a multiple random coupling mechanism. More than one lipoyl moiety services each E1 subunit (alpha-ketoglutarate dehydrogenase, EC 1.2.4.2), and an extensive lipoyl-lipoyl interaction network for exchange of electrons and possibly acyl groups must also be present. The best fit between computed and experimental data was obtained with a model that has two lipoyl moieties servicing each E1 subunit and a lipoyl-lipoyl interaction network that links all lipoyl moieties on the E2 cube (dihydrolipoamide succinyltransferase, EC 2.3.1.61). The single lipoyl moiety on an E2 subunit is assumed to service the coenzyme A-dependent succinyltransferase site of that E2 subunit as well as an E3 subunit (dihydrolipoamide dehydrogenase, EC 1.6.4.3) if the latter is bound to that particular E2 subunit.     

Molecular Structure of the Pyruvate Dehydrogenase Complex from Escherichia coli K-12

The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex that can be obtained with a unique polypeptide chain composition, has a molecular weight of 3.75 x 10(6). All results obtained agree with the following numerology. The core complex consists of 48 polypeptide chains. There are 16 chains (molecular weight = 100,000) of the pyruvate dehydrogenase component, 16 chains (molecular weight = 80,000) of the dihydrolipoamide dehydrogenase component, and 16 chains (molecular weight = 56,000) of the dihydrolipoamide dehydrogenase component. Usually, but not always, pyruvate dehydrogenase complex is produced in vivo containing at least 2-3 mol more of dimers of the pyruvate dehydrogenase component than the stoichiometric ratio with respect to the core complex. This "excess" component is bound differently than are the eight dimers in the core complex.     

Reactivity of primary biliary cirrhosis sera with Escherichia coli dihydrolipoamide acetyltransferase (E2p): characterization of the main immunogenic region.

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by the presence of antimitochondrial autoantibodies in the serum. The major antigens recognized by the antibodies are the E2 components of the 2-oxo acid dehydrogenase complexes, all of which possess covalently attached lipoic acid cofactors. A bacterial etiology has been proposed for the disease, and patients' antibodies are known to recognize the E2 subunits (E2p) of both mammalian and bacterial pyruvate dehydrogenase complexes. Immunoblotting and ELISA inhibition techniques using extracts of Escherichia coli deletion strains, genetically restructured E2 polypeptides, and isolated lipoyl domains demonstrate that (i) the E2o subunit of the E. coli 2-oxoglutarate dehydrogenase complex is recognized by patients' antibodies; (ii) the main immunogenic region of E2p lies within the lipoly domains; (iii) the presence of a lipoly residue within the domain is crucial for effective recognition by the antibodies; and (iv) octanoylated E2p, octanoylated E2o, and octanoylated lipoyl domain, produced by a mutant deficient in lipoate biosynthesis, are recognized by patients' antibodies but not as effectively as their lipoylated counterparts. These findings indicate that antibodies in PBC patients' sera bind to a unique peptide-cofactor conformation within the lipoyl domains of the E2 polypeptides and that this epitope is partially mimicked by substituting the lipoyl cofactor with an octanoyl group.     

Nucleotide sequence for yeast dihydrolipoamide dehydrogenase.

Rabbit antiserum to the dihydrolipoamide dehydrogenase (dihydrolipoamide:NAD+ oxidoreductase, EC 1.8.1.4) component of the pyruvate dehydrogenase complex from bakers' yeast was used to screen plaques produced by a lambda gt11 yeast cDNA library. A 2.1-kilobase insert was isolated that also hybridized to a 17-base mixed oligonucleotide probe corresponding to the amino-terminal sequence of the yeast dihydrolipoamide dehydrogenase. The cDNA has a coding sequence of 499 amino acids that corresponds to a 21-residue signal peptide and a 478-residue mature protein (Mr = 51,558). Computer analysis shows that yeast dihydrolipoamide dehydrogenase has about 41% amino acid identity with Escherichia coli dihydrolipoamide dehydrogenase. Particularly striking is the conservation of sequence in the active site region of the dihydrolipoamide dehydrogenases from E. coli, yeast, and pig heart.     

3D electron microscopy reveals the variable deposition and protein dynamics of the peripheral pyruvate dehydrogenase component about the core

Cryo-electron microscopy was exploited to reveal and study the influence of pyruvate dehydrogenase (E1) occupancy on the conformational states of the Saccharomyces cerevisiae pyruvate dehydrogenase complex (PDC). Structures representative of PDC preparations with approximately 40% and full E1 occupancy were determined after the electron microscopy images from each preparation were classified according to their sizes. The reconstructions derived from two size groups showed that the deposition of the E1 molecules associated with the larger complex is, unexpectedly, not icosahedrally arranged, whereas in the smaller complex the E1 molecules have an arrangement and architecture similar to their more ordered deposition in the WT bovine kidney PDC. This study also shows that the linker of dihydrolipamide acetyltransferase (E2) that tethers E1 to the E2 core increases in length from approximately 50 to 75 A, accounting largely for the size difference of the smaller and larger structures, respectively. Extensive E1 occupancy of its 60 E2 binding sites favors the extended conformation of the linker associated with the larger complex and appears to be related to the loss of icosahedral symmetry of the E1 molecules. However, the presence of a significant fraction of larger molecules also in the WT PDC preparation with low E1 occupancy indicates that the conformational variability of the linker contributes to the overall protein dynamics of the PDC and the variable deposition of E1. The flexibility of the complex may enhance the catalytic proficiency of this macromolecular machine by promoting the channeling of the intermediates of catalysis between the active sites.     

Identification and analysis of the major M2 autoantigens in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by the presence of antimitochondrial antibodies in the serum. It is possible that the PBC-specific immunoreactive trypsin-sensitive antigens on the inner mitochondrial membrane, termed M2, are important in the pathogenesis of this autoimmune disease. We have previously shown that a major M2"a" antigen is the E2 component of the pyruvate dehydrogenase multienzyme complex located within mitochondria. Analysis of the primary structure of the E2 components of all three 2-oxo acid dehydrogenase complexes reveals a high degree of homology with a similar highly segmented structure including lipoyl domains, E3-binding domains, C-terminal catalytic domains, and interdomain linker sequences. Immunoblotting of PBC patients' sera against purified E2 protein from 2-oxoglutarate dehydrogenase complex and branched-chain 2-oxo acid dehydrogenase complex reveals that these polypeptides are also autoantigens in this disease. Sera from 29 of 40 (72.5%) PBC patients gave a positive response against bovine 2-oxoglutarate dehydrogenase complex E2 and from 25 of 40 (62.5%) PBC patients gave a positive response against bovine branched-chain 2-oxo acid dehydrogenase complex E2. All 40 PBC patients (100%) have autoantibodies directed against at least one of the E2 components of the family of 2-oxo acid dehydrogenase complexes. Identification of these M2 mitochondrial autoantigens and detailed knowledge of their structure will allow important questions concerning this autoimmune disease to be addressed.     

Epitope mapping on E1α subunit of pyruvate dehydrogenase complex with autoantibodies of patients with primary biliary cirrhosis

Background: A major mitochondrial autoantigen recognized by sera of patients with primary biliary cirrhosis (PBC) is dihydrolipoamide acetyltransferase (E2) of the pyruvate dehydrogenase complex (PDH). The α subunit of pyruvate decarboxylase (E1α) of PDH is also recognized in some E2‐reactive PBC sera, suggesting that the occurrence of autoimmunity against E1α is subsequent to that against E2. Methods: To investigate the mechanism inducing autoimmunity against E1α, we surveyed immunoreactive sequences of E1α by ELISA with synthesized oligopeptides, and determined minimum amino acid residues for each determinant. Results: The major determinants of E1α appeared to reside in its N‐terminal region, apparently forming ‘nested epitopes’, and all E1α‐reactive PBC sera tested recognized these regions. Minor epitopes were also found scattered throughout the entire sequence. The reactivities of these minor epitopes to individual PBC sera were proportional to those of the major epitopes. All the epitopes were located in hydrophilic regions of E1α, and many of them were out of the known functional domains (TPP‐binding domain, subunit interaction site, and phosphorylation sites) whose structures are phylogenically well conserved. Furthermore, the sequences of many epitopes appeared to be specific to humans. Conclusion: These observations suggest that determinant spreading might underlie the autoimmunity against E1α.     

Cloning and nucleotide sequence of the gene for protein X from Saccharomyces cerevisiae.

The gene encoding the protein X component of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae has been cloned and sequenced. A 487-base fragment of yeast genomic DNA encoding the amino-terminal region of protein X was amplified by the polymerase chain reaction using synthetic oligonucleotide primers based on amino-terminal and internal amino acid sequences. This DNA fragment was used as a probe to select two genomic DNA restriction fragments, which were cloned and sequenced. A 2.1-kilobase insert contains the complete sequence of the protein X gene. This insert has an open reading frame of 1230 nucleotides encoding a presequence of 30 amino acid residues and a mature protein of 380 amino acid residues (Mr, 42,052). Hybridization analysis showed that there is a single copy of the protein X gene and that the size of the mRNA is approximately 1.5 kilobases. Comparison of the deduced amino acid sequences of yeast protein X and dihydrolipoamide acetyltransferase indicates that the two proteins evolved from a common ancestor. The amino-terminal part of protein X (residues 1-195) resembles the acetyltransferase, but the remainder is quite different. There is strong homology between protein X and the acetyltransferase in the amino-terminal region (residues 1-84) that corresponds to the putative lipoyl domain. Protein X lacks the highly conserved sequence His-Xaa-Xaa-Xaa-Asp-Gly near the carboxyl terminus, which is thought to be part of the active site of all dihydrolipoamide acyltransferases.     

Subunit structure of dihydrolipoyl transacetylase component of pyruvate dehydrogenase complex from Escherichia coli

Limited tryptic digestion of the pyruvate dehydrogenase complex of Escherichia coli or its dihydrolipoyl transacetylase core cleaves the trypsin-sensitive transacetylase subunits into two large fragments, A (lipoyl domain) and D (subunit binding domain). Release of fragments A from the complex does not significantly affect its sedimentation coefficient or its appearance in the electron microscope. Fragment A contains the lipoyl moieties ((3)H-labeled), is acidic with an apparent isoelectric point of about 4.0, has a M(r) of 31,600 as determined by sedimentation equilibrium analysis, and has a swollen or extended structure (f/f(o) = 1.78). Fragment A exhibits anomalous properties, probably due to its acidic nature. It is resistant to staining with Coomassie blue and it migrates on sodium dodecyl sulfate/polyacrylamide gels as if it had a M(r) of 46,000-48,000. Further tryptic digestion converts fragment A into a lipoyl-containing fragment of M(r) 20,000 (fragment B) and eventually into an apparently stable product of estimated M(r) about 10,000 (fragment C). Fragment D has a compact structure of M(r) about 29,600 as determined by sedimentation equilibrium analysis in 6 M guanidinium chloride, and it possesses the intersubunit binding sites of the transacetylase, the binding sites for pyruvate dehydrogenase and dihydrolipoyl dehydrogenase, and the catalytic site for transacetylation. The assemblage of fragments D is responsible for the cube-like appearance of the transacetylase in the electron microscope. High-resolution electron micrographs of the transacetylase show fiber-like extensions, apparently corresponding to tryptic fragment A, surrounding the cube-like core.     

Autoantibodies in primary biliary cirrhosis: analysis of reactivity against eukaryotic and prokaryotic 2-oxo acid dehydrogenase complexes

Six components of the mammalian 2-oxo acid dehydrogenase complexes have previously been identified as M2 autoantigens in primary biliary cirrhosis. In this report, we present data showing that both polypeptide-specific and cross-reacting antibodies are present in patients' sera. Antibodies reacting with E2 of the pyruvate dehydrogenase complex cross-react with protein X but not with any other mammalian antigen. The main immunogenic region on protein X has been localized to within its single lipoyl domain. Polypeptide-specific antibodies bind to E1 alpha and E1 beta of the pyruvate dehydrogenase complex. Antibodies reacting with the E2 polypeptides of the 2-oxoglutarate dehydrogenase complex and branched-chain 2-oxo acid dehydrogenase complex show some cross-reactivity but do not recognize any of the antigens of the pyruvate dehydrogenase complex. Antibodies against the E2 component of the mammalian pyruvate dehydrogenase complex cross-react effectively with the corresponding protein from yeast but not with E2 from Escherichia coli. Antibody titer against mammalian antigens is significantly higher than against the bacterial antigens, arguing against a bacterial origin for primary biliary cirrhosis.     

Actinobacteria challenge the paradigm: A unique protein architecture for a well-known,central metabolic complex

α-oxoacid dehydrogenase complexes are large, tripartite enzymatic machineries carrying out key reactions in central metabolism. Extremely conserved across the tree of life, they have been, so far, all considered to be structured around a high–molecular weight hollow core, consisting of up to 60 subunits of the acyltransferase component. We provide here evidence that Actinobacteria break the rule by possessing an acetyltranferase component reduced to its minimally active, trimeric unit, characterized by a unique C-terminal helix bearing an actinobacterial specific insertion that precludes larger protein oligomerization. This particular feature, together with the presence of an odhA gene coding for both the decarboxylase and the acyltransferase domains on the same polypetide, is spread over Actinobacteria and reflects the association of PDH and ODH into a single physical complex. Considering the central role of the pyruvate and 2-oxoglutarate nodes in central metabolism, our findings pave the way to both therapeutic and metabolic engineering applications.

The α-oxoacid dehydrogenase complexes constitute a family of three-partite, ubiquitous metabolic complexes devoted to the oxidative decarboxylation of α-oxoacids and the concomitant production of reducing equivalents in form of NADH (1). Three such complexes are known: the pyruvate dehydrogenase (PDH), that feeds the tricarboxylic acid (TCA) cycle with carbon units in form of acetyl-CoA; the 2-oxoglutarate dehydrogenase (ODH), part of the oxidative branch of the TCA cycle; and the branched chain α-ketoacid dehydrogenase (BCKDH), involved in the catabolism of aliphatic amino acids. These large tripartite complexes share a common molecular architecture organized around a core made by the E2 component, a flexible, multidomain protein which bears the acyltransferase activity required to transfer the acyl group from the decarboxylated substrate to the CoA-SH acceptor; the number of E2 subunits and the symmetry of the core depend on the complex and the species (1–5). First shown by the crystal structure of Azotobacter vinelandii E2p, the E2 C-terminal catalytic core assumes an obligate homotrimeric state much similar to chloramphenicol acetyltransferase (6), with which it also shares the catalytic mechanism (7). The observed, higher-order oligomerization states are made possible by intermolecular trimer–trimer interactions (TTIs) mediated by a well-conserved, C-terminal 3₁₀ hydrophobic helix which makes intermolecular symmetric interactions (5), then confirmed on other E2 enzymes and sometimes described as “knobs and sockets” (3). These interactions make symmetric, highly oligomeric states which adopt, in most cases, either an octahedral 432 symmetry, eight E2 homotrimers being positioned at the vertexes of a cube, or an icosahedral 532 symmetry, with 20 trimers assembled as a dodecahedron; the number of subunits depends on the complex and the species (1). More recently, the presence of an irregularly shaped, 42-mer E2 assembly was described in the archaeon Thermoplasma acidophilum (8), although this peculiar oligomeric state is still based on the same kind of interactions between the C-terminal helices. Thus, the oligomeric state of the core, responsible for the large size of the complex, was observed in all analyzed complexes and is a trend commonly accepted to be universally conserved in Eubacteria, Archaea, and Eukarya. While the reasons for the presence (and evolutionary conservation) of such huge macromolecular scaffolds remain unclear (9), active site coupling (transfer of acyl groups between lipoyl domains within the core) has often been proposed as the major advantage (1, 10). Also, despite the tripartite organization of these complexes as separate E1/E2/E3 enzymes had always been considered as universal, Corynebacterium glutamicum was shown to be deprived of an E2o component (specific to the ODH complex) and to rather possess an E2o succinyl transferase domain fused to E1o, in a protein called OdhA (11, 12). The same situation has then been confirmed for the model Mycobacterium smegmatis (13). As the lipoyl binding domain of E2o is absent from the E2o-E1o fusion, the ODH activity depends on functional lipoyl groups provided in trans and proven to be supplied by E2p from the PDH complex (14), therefore suggesting the presence of a mixed PDH/ODH supercomplex. By using an integrative structural biology approach, we describe here how C. glutamicum E2p, that was expected to serve as the core of the mixed complex, breaks the rule about the oligomeric state of acyltransferase E2 enzymes, reducing its size to the minimal, catalytically active trimeric unit. We also provide evidence supporting these features as a common trait of Actinobacteria.     

Cloning and nucleotide sequence of the gene for dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae.

A 537-base cDNA encoding a portion of Saccharomyces cerevisiae dihydrolipoamide acetyltransferase (acetyl-CoA:dihydrolipoamide S-acetyltransferase, EC 2.3.1.12) was isolated from a lambda gt11 yeast cDNA library by immunoscreening. This cDNA was subcloned and used as a probe to screen a lambda gt11 yeast genomic DNA library. Two overlapping clones were used to determine the complete sequence of the acetyltransferase gene. The composite sequence has an open reading frame of 1446 nucleotides encoding a presequence of 28 amino acids and a mature protein of 454 amino acids (Mr = 48,546). The deduced amino acid sequence contains the experimentally determined amino acid sequences of the amino terminus and two internal peptide fragments of the acetyltransferase. Hybridization analysis of yeast genomic DNA showed that the gene has a single copy. A 915-base segment of the acetyltransferase gene hybridized to a yeast mRNA of approximately equal to 1.6 kilobases. Analysis of the deduced amino acid sequence of the dihydrolipoamide acetyltransferase revealed a multidomain structure similar to those reported for the corresponding acetyltransferases from Escherichia coli and rat liver, and extensive sequence similarity among the three enzymes. However, the yeast enzyme contains only one lipoyl domain, in contrast to three lipoyl domains reported for the E. coli enzyme and apparently two for the rat liver enzyme.     

Reconstitution of the Escherichia coli pyruvate dehydrogenase complex.

The binding of pyruvate dehydrogenase and dihydrolipoyl dehydrogenase (flavoprotein) to dihydrolipoyl transacetylase, the core enzyme of the E. coli pyruvate dehydrogenase complex [EC 1.2.4.1:pyruvate:lipoate oxidoreductase (decaryboxylating and acceptor-acetylating)], has been studied using sedimentation equilibrium analysis and radioactive enzymes in conjunction with gel filtration chromatography. The results show that the transacetylase, which consists of 24 apparently identical polypeptide chains organized into a cube-like structure, has the potential to bind 24 pyruvate dehydrogenase dimers in the absence of flavoprotein and 24 flavoprotein dimers in the absence of pyruvate dehydrogenase. The results of reconstitution experiments, utilizing binding and activity measurements, indicate that the transacetylase can accommodate a total of only about 12 pyruvate dehydrogenase dimers and six flavoprotein dimers and that this stoichiometry, which is the same as that of the native pyruvate dehydrogenase complex, produces maximum activity. It appears that steric hindrance between the relatively bulky pyruvate dehydrogenase and flavoprotein molecules prevents the transacetylase from binding 24 molecules of each ligand. A structural model for the native and reconstituted pyruvate dehydrogenase complexes is proposed in which the 12 pyruvate dehydrogenase dimers are distributed symmetrically on the 12 edges of the transacetylase cube and the six flavoprotein dimers are distributed in the six faces of the cube.     

Frequency of IgG and IgM autoantibodies to four specific M2 mitochondrial autoantigens in primary biliary cirrhosis

We have previously identified four of the M2 antigens in primary biliary cirrhosis as the E2 components (dihydrolipoamide acyltransferases) of pyruvate dehydrogenase complex, branched-chain 2-oxo acid dehydrogenase complex and 2-oxoglutarate dehydrogenase complex and the protein X component of pyruvate dehydrogenase complex (approximate molecular masses: 74, 50, 50 and 52 kD, respectively). In the present study, we have examined by immunoblotting the frequency of IgG and IgM autoantibodies to these four proteins in 129 patients with primary biliary cirrhosis (36 histological Stage I, 42 Stage II/III, 51 Stage IV) and 77 controls (49 non-primary biliary cirrhosis chronic liver disease, 16 primary Sj?gren's syndrome, 12 healthy normal women). One hundred twenty-seven of 129 (98%) primary biliary cirrhosis patients had antibodies against at least one of the four M2 polypeptides, compared to 2/77 controls (both had autoimmune chronic active hepatitis and were antimitochondrial antibody positive by indirect immunofluorescence). One hundred twenty-one of 129 (94%) primary biliary cirrhosis sera reacted with the E2 component and protein X of pyruvate dehydrogenase complex, 69/129 (53%) primary biliary cirrhosis sera reacted with E2 of branched-chain 2-oxo acid dehydrogenase complex and 113/129 (88%) reacted with E2 of 2-oxoglutarate dehydrogenase complex. Primary biliary cirrhosis patients with histological Stage I disease had a lower incidence of autoantibodies to each M2 protein, compared to more advanced disease (IgG, p less than 0.05) but only 2/36 Stage I patients had no anti-M2 antibodies. There was no correlation between the presence of IgG or IgM antibodies to the M2 polypeptides and established prognostic markers in primary biliary cirrhosis (serum bilirubin and albumin levels).(ABSTRACT TRUNCATED AT 250 WORDS)     

Lactic acidosis and developmental delay due to deficiency of E3 binding protein (protein X) of the pyruvate dehydrogenase complex

Pyruvate dehydrogenase deficiency is an important cause of primary lactic acidosis. Most cases occur as a result of mutations in the gene for the E1 alpha subunit of the complex, with a small number resulting from mutations in genes for other components, most commonly the E3 and E3-binding protein subunits. We describe pyruvate dehydrogenase E3-binding protein deficiency in two siblings in each of two unrelated families from Kuwait. The index patient in each family had reduced pyruvate dehydrogenase activity in cultured fibroblasts and no detectable immunoreactive E3-binding protein. Both were homozygous for nonsense mutations in the E3-binding protein gene, one involving the codon for glutamine 266, the other the codon for tryptophan 5.     

Catalytic domain of PDC-E2 contains epitopes recognized by antimitochondrial antibodies in primary biliary cirrhosis

AIM:To search for further immunodominant peptides of the pyruvate dehydrogenase complex E2-component (PDC-E2) recognized by antimitochondrial antibodies (AMA) in primary biliary cirrhosis (PBC). METHODS:Sera from 95 patients with PBC were tested by enzyme-linked immunosorbent assay against 33 synthetic overlapping peptides (25 amino acids; aa) covering the entire length of the E2-subunit of PDC-E2. Furthermore,the inner lipoyl peptide 167-184 was used in an unlip oylated and a lipoylated form as well as cou...     

Rapid intramolecular coupling of active sites in the pyruvate dehydrogenase complex of Escherichia coli: Mechanism for rate enhancement in a multimeric structure

In the absence of CoA and presence of pyruvate, the lipoic acid residues covalently bound to the lipoate acetyltransferase core component (acetyl-CoA:dihydrolipoate S-acetyltransferase, EC 2.3.1.12) of the pyruvate dehydrogenase multienzyme complex of Escherichia coli become reductively acetylated. A study of a series of reassembled complexes varying only in their content of pyruvate decarboxylase [pyruvate:lipoate-oxidoreductase (decarboxylating and acceptor-acetylating) EC 1.2.4.1] showed that the initial direct reductive acetylation of lipoic acid residues can be followed by extensive intramolecular transacetylation reaction between lipoic acid residues on neighboring polypeptide chains of the lipoate acetyltransferase core [Bates, D. L., Danson, M. J., Hale, G., Hooper, E. A. & Perham, R. N. (1977) Nature (London) 268, 313-316]. Pulsed-quenched-flow measurements of the rates of the acetylation reactions in the various complexes now demonstrate that the intramolecular transacetylation reactions are not rate-determining in the normal reaction mechanism of the enzyme. There is therefore the potential for rapid multiple coupling of active sites in the lipoate acetyltransferase core. The rate constant for the overall complex reaction, measured by stopped-flow fluorimetry, is found to be approximately twice that for the reductive acetylation reaction measured by pulsed-quenched flow. This result could mean that CoA is an allosteric stimulator of the reductive acetylation part of the overall reaction or that there are two active sites on each chain of the lipoate acetyltransferase component working in parallel. A system of rapid functional connection of active sites in a multienzyme complex ensures that sequential reactions can be successfully coupled even under conditions of low substrate concentrations for the different steps. The substantial rate enhancement thus achieved offers a plausible explanation for the unusual complexity of the quaternary structure of the enzyme.     

Molecular mimicry of mitochondrial and nuclear autoantigens in primary biliary cirrhosis

BACKGROUND & AIMS: The mechanism for development of primary biliary cirrhosis (PBC) remains enigmatic, but molecular mimicry has been implicated because of well-known cross-reactivity of human mitochondrial autoantigens and equivalent bacterial antigens. Virtually all patients with PBC have antimitochondrial autoantibodies (AMA), but, interestingly, approximately 50% also manifest antinuclear antibodies (ANA). METHODS: To determine whether generation of ANA are due to molecular mimicry of mitochondrial peptides, we established 6 T-cell clones selected by a peptide corresponding to the E2 subunit of mitochondrial pyruvate dehydrogenase complex and analyzed for reactivity to mimicry peptides derived from mitochondrial and nuclear autoantigens, including control sequences. RESULTS: For mitochondrial autoantigens, 1 peptide from the E2 subunit of the pyruvate dehydrogenase complex, 1 peptide from the E2 subunit of the oxo-glutarate dehydrogenase complex, 1 peptide from the E2 subunit of the branched-chain 2-oxoacid dehydrogenase complex, and 1 peptide from the E3-binding protein cross-reacted with these T-cell clones. For the nuclear autoantigens, 5 peptides from gp210 and 1 from Sp100 cross-reacted with these clones. Furthermore, 1 of 3 T-cell clones selected by recombinant gp210 protein reacted with a mimicry peptide corresponding to amino acids 188-201 of gp210, indicating that this part of the protein is a naturally processed immunodominant T-cell epitope. CONCLUSIONS: These results demonstrate molecular mimicry between mitochondrial and nuclear autoantigens in PBC and that a mimicry peptide may become an immunodominant T-cell epitope. These data have significance not only for PBC but also for the production of ANA in other disease processes.