The M_r‐50000 polypeptide of mammalian pyruvate dehydrogenase complex participates in the acetylation reactions

Abstract

The mammalian pyruvate dehydrogenase complex, M , 8.5 x lo", contains an additional tightly bound 50 000-M , polypeptide, Component X, which copurifies with the intact assembly.Small amounts of the individual E2 and X polypeptides were obtained by elution of the protein bands from SDS/polyacrylamide gels. One-dimensional peptide mapping studies with ' 251-labelled lipoyl acetyltransferase (E2) and component X subunits indicate that these two proteins are structurally distinct entities. Similar analysis of purified subunits, initially radiolabelled in the intact complex in the presence of [2-14C]pyruvate and N-ethyl-[2,3-*4C]maleimide confirm that distinct 14C-labelled peptides are generated from these two species. These proteinchemical data supplement recent immunological findings, which demonstrate that component X is not a proteolytic fragment of the larger lipoyl acetyltransferase ( M , 70000) subunit.Incubation of the native PDC in the presence of [2-14C]pyruvate leads to rapid uptake of radiolabel, presumably as acetyl groups, into both E2 and protein X. Specific incorporation of acetyl groups declines to a similar extent on both polypeptides after inhibiting pyruvate dehydrogenase (El) activity by phosphorylation or omitting thiamine diphosphate (TPP) from the assay mixture. Addition of CoASH promotes the parallel deacetyhtion of both lipoyl acetyltransferase and protein X in a reaction which displays sensitivity to N-ethylmaleimide.The unique structural, enzymological and regulatory features of the mammalian pyruvate dehydrogenase complex (PDC) have prompted detailed investigation of its properties over many years [I, 21. The macromolecular array of three individual enzymes is responsible for controlling the supply of CoASAc to the tricarboxylic acid cycle by catalysing its formation from pyruvate. Glucose is conserved in mammals in starvation and diabetes by diminished flow through the mitochondria1 PDC [3]. Its overall activity is regulated by a phosphorylation/dephosphorylation system involving a specific pyruvate dehydrogenase kinase and a phosphatase, with phosphorylation resulting in inactivation [I 1.The major enzymatic activities, catalyzing discrete partial reactions of the complete process, have long been recognised as they can be purified readily in active form after dissociation of the complex under mild conditions [4, 51. The initial step involves decarboxylation of the substrate by pyruvate dehydrogenase (El), a TPP-requiring a2/32 tetramer, which also transfers two carbon units to lipoate residues, situated on lipoyl acetyltransferase (E2), in a reductive acetylation reaccorrespondence to

Defects in the E2 lipoyl transacetylase and the X-lipoyl containing component of the pyruvate dehydrogenase complex in patients with lactic acidemia.

“…Interestingly, the abnormality present in E2 affects measurable transacetylase activity but those in protein X do not. This suggests that X itself does not contribute significantly to the cellular dihydrolipoyl transacetylase activity despite the fact that the lipoyl groups on X can be acetylated (1,2). Ifthe PDH complex could be purified from patient derived cell lines in sufficient quantity to allow detailed study more information could be gained about the role ofthe two lipoyl proteins in the complex.…”

“…This protein, whose mobility on sodium dodecyl sulfate polyacrylamide gels is slightly greater than the E3 component lipoamide dehydrogenase (Mr = 53,000), resembles the E2 dihydrolipoyl tranacetylase protein in that it bears a lipoyl group attached to a lysine residue (3,4). This lipoyl residue is capable of being acetylated when the complex is treated with pyruvate (1,2), though protein X itself has no innate dihydrolipoyl transacetylase activity. The study of human pyruvate dehydrogenase complex deficiency has shown that defects in the El compo-nent are most common, with a smaller number of E3 defects (5).…”

“…The mammalian pyruvate dehydrogenase complex (PDHC)' differs from the complex in Escherichia coli and from the other mammalian a-keto-acid hydrogenases by the presence of a 53-kD protein that has been labeled protein X (1,2). This protein, whose mobility on sodium dodecyl sulfate polyacrylamide gels is slightly greater than the E3 component lipoamide dehydrogenase (Mr = 53,000), resembles the E2 dihydrolipoyl tranacetylase protein in that it bears a lipoyl group attached to a lysine residue (3,4).…”

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

“…Both E2 and protein X participate in the acetylation reactions of the complex [6] and apparently act as independent substrates for reductive acetylation by pyruvate dehydrogenase (El) [23]. The inability to dissociate protein X from the E2 core assembly except under denaturing conditions also highlights the close physical and functional integration of these two components [3].…”

“…In the native complex, 20-30 ce2B2 tetramers of pyruvate dehydrogenase, E1 (EC 1.2.4.1) and 6 homodimers of dihydrolipoamide dehydrogenase, E3 (EC 1.8.1.4) are attached non-covalently to a multimeric core assembly of 60 lipoic acid-containing acetyltransferase (E2) subunits (EC 2.3.1.12), organised in the form of a pentagonal dodecahedron [1,2]. Attention has also focused on a minor core species, protein or component X [3,4], a distinct lipoylbearing polypeptide [5] which is tightly-bound to E2 and also participates in the acetylation reactions of the complex [6].…”