Lipoic acid is the site of substrate‐dependent acetylation of component X in ox heart pyruvate dehydrogenase multienzyme complex

Abstract

The recently characterized M,-50000 polypeptide associated with mammalian pyruvate dehydrogenase complex, referred to as component or protein X, was shown to incorporate N-ethylmaleimide only in the presence of pyruvate or NADH. Component X, modified with N-ethy1[2,3-'4C]maleimide in the presence of pyruvate. was isolated and subjected to acid hydrolysis. The radioactive products were resolved on an amino acid analyser and these coeluted with products from similarly modified and hydrolysed lipoate acetyltransferase. Preincubation of pyruvate dehydrogenase complex with pyruvate or NADH and acetyl-CoA resulted in a time-dependent diminution of incorporation of radiolabelled N-ethylmaleimide into component X and lipoate acetyltransferase and, correspondingly, in the extent of inhibition of overall complex activity by N-ethylmaleimide.Pyruvate dehydrogenase complex (PDC) from mammalian sources comprises a structural core formed by 60 E2 subunits ( M , 52000) organised into a pentagonal dodecahedron (532 symmetry), to which El( a2p2 = M , 154000) and E3 ( a homodimer of M , 110000) subunits are bound to give a large array of 8.5 x lo6 [2]. Most evidence suggests that only a single lipoyl moiety is present on each E2 polypeptide [2, 31, although there have been some contrary indications [4]. Lipoic acid is covalently attached to E2 polypeptides by means of an amide bond to the Nb-amino group of lysine residues [5]. Studies employing NMR or electron microscopy [6, 71 have shown that the lipoyl groups of E2 subunits are sited on extended regions of polypeptide, which are highly mobile relative to other parts of the multienzyme complex. This would enable any one lipoyl group to interact over more of the complex surface than the length of the lysine-lipoate arm, 1.4 nm, would at first suggest. Limited proteolysis of ox kidney PDC results in the release of a domain bearing lipoic acid from the E2 subunit leaving a structural domain which remains as a high-M, assemblage [8]. Substrate-induced S-acetylation of lipoyl groups on the E2 subunits generates an extra thiol on the adjacent sulphur atom in the dithiolane ring, which may thus be specifically modified with N-ethylmaleimide [9].It has become clear recently that mammalian PDC contains a previously unrecognised M , 50000 polypeptide, which forms a close physical and functional union with the E2 assembly [lo]. In addition the E2 assembly, isolated from the PDC of Saccharomyces cerevisiae, shows a tightly bound polypeptide of approximate M , 50000, which is in similar

Characterization of the autoantibody responses to recombinant E3 binding protein (protein X) of pyruvate dehydrogenase in primary biliary cirrhosis

“…23 It is also now well established that the human antibody response to PDC-E2 is almost exclusively directly against the inner lipoic acid-containing domain of the protein, 24 the structure of which has recently been determined. 25 The lipoic acid molecule itself seemingly forms part of the B-cell epitope.…”

Lipoic Acid Metabolism in Microbial Pathogens

“…The E3-binding protein (E3BP) is homologous to E2 subunits and includes a single lipoyl domain followed by a peripheral-subunit-binding domain (PSBD) and the catalytic domain (77,155). The lipoyl domain is lipoylated and can be reduced and acetylated by the E3 and E1 subunits of PDH (85,100,181). However, E3BPs do not seem to catalyze the transacetylase reaction necessary to generate acetyl-CoA, perhaps due to the absence of a catalytic histidine residue which is present in E2 subunits (77).…”

Organization of the Cores of the Mammalian Pyruvate Dehydrogenase Complex Formed by E2 and E2 Plus the E3-binding Protein and Their Capacities to Bind the E1 and E3 Components

“…Besides those universal components, the mammalian and subsequently some other eukaryotic PDCs were shown to contain another component, called E3-binding protein (E3BP); this protein was originally designated protein X (6, 7). Mammalian E3BP was first characterized as a component with a reactive lipoyl group on a single lipoyl domain (7)(8)(9)(10)(11) that, alone, could support the overall reaction (12). E3BP was tightly bound to E2 (7) by its C-terminal region (10).…”