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