Structure function studies on the lipoate‐acetyltransferase–component‐X‐core assembly of the ox heart pyruvate dehydrogenase complex

Abstract

Component X, the recently recognised subunit of mammalian pyruvate dehydrogenase complex, was shown by immune blotting to be present in all of nine tissues dissected from rat. This finding indicated that component X was not an isoenzyme of the lipoate acetyltransferase (E2) associated with one or a limited number of tissues.Native pyruvate dehydrogenase complex was shown to bind IgG raised to isolated component X, indicating that there were at least some regions of the X subunit exposed at the periphery of the complex.Lipoyl groups of ox heart pyruvate dehydrogenase complex were specifically cross-linked by reaction with phenylene-o-bismaleimide in the presence of pyruvate and the subunits contributing to the products of crosslinking were identified by immune blotting. Species with very high MI containing both E2 and component X, were formed in high yield, as well as apparent E2/E2 and E2/X dimers and trimers and an X/X dimer. These results showed that acetylated lipoyl groups of different E2 and X subunits were able to interact in all possible combinations.The types of cross-linked E2 products formed suggested that two thiols, reactible with phenylene-o-bismaleimide, were rapidly generated in the presence of pyruvate. The results were most easily explained by the presence of two acetylatable lipoyl groups on each E2 polypeptide.Pyruvate dehydrogenase complex (PDC) is responsible for the conversion of pyruvate to acetyl-CoA with the overall reactionIn mammalian systems the enzyme is located in the mitochondrion and is a large multienzyme complex of approximate M I = 8.5 x lo6 formed by an assembly of 60 lipoate acetyltransferase subunits (E2) arranged as a pentagonal dodecahedron, bound to which are multiple copies of pyruvate dehydrogenase (El ) and lipoamide dehydrogenase (E3) subunits [l]. Also bound to the pyruvate dehydrogenase complex, although in lower proportions, is a specific kinase involved in regulation of the complex activity by means of covalent modification. The complex is inactivated by the intrinsic kinase, which phosphorylates the Ela subunit and reactivation is achieved by the action of pyruvate dehydrogenase phosphatase [2]. The phosphatase activity does not copurify with the complex. An additional subunit of MI 50000 has recently been identified and has been designated protein or component X [3,4]. Similarly to E2, component X contains covalently bound lipoic acid [5] which is acetylated in the presence of pyruvate or acetyl-CoA and may be reduced by NADH [6]. Immunological characterisation and comparison of peptides produced by limited proteolysis has indicated that component X is unlikely to be derived from E2 by proteolytic cleavage of the E2 polypeptide [3, 61.Studies employing electron microscopy have shown that the lipoyl groups on E2 subunits of PDC are located on peripherally extended regions of polypeptide [7] and analysis with proton NMR show these to be highly mobile relative to other parts of the complex [S]. This mobility would allow interaction of the lipoic acid at spatially...

Antimitochondrial autoantibodies in primary biliary cirrhosis recognize cross-reactive epitope(s) on protein X and dihydrolipoamide acetyltransferase of pyruvate dehydrogenase complex

“…in Mitochondria from Other Species. It has been documented that protein X is present in mitochondria from a variety of mammalian species beside bovine mitochondria, including pig and rat (15,27). However, we previously have not observed an AMA-speci fic antigen of 52 to 56 kD molecular weight, other than the 50 to 52 kD BCKD-E2, in mitochondria other than those of bovine origin (3).…”

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

“…Hence, all 40 PBC patients had autoantibodies directed against at least one of the E2 components of the family of 2-oxo acid dehydrogenase complexes, with some sera reacting with one, two, or three E2s (Table 1 (37,38) rather than the E. coli PDC E2, which has three lipoyllysine residues per E2. The presence oftwo (or more) lipoate residues in mammalian PDC E2 has also been strongly suggested from recent crosslinking studies (39). The reason for having more than one lipoyl domain is unclear because genetically engineered derivatives of the E2p component of E. coli having only one or two lipoyl domains per chain appear to be fully active (40), and it has long been known that up to half of the lipoyl groups 8658 Medical Sciences: Fussey et al…”

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

“…125 amino acid lipoyl domain from protein X. These domains are known to migrate anomalously on SDS-polyacrylamide gels owing to the presence of Ala-Pro-rich linker regions [11]; and (iii) specific cross-linking studies on E2 and X with phenylene-o-bismaleimide via substrate-generated thiols on lipoic acid groups has provided evidence for the presence of two lipoyl groups per E2 chain and only a single lipoate on protein X [9].…”

“…Recent cloning and sequence analysis of the human lipoate acetyltransferase (E2) gene [7,8] in conjunction with earlier protein-chemical studies [9] have demonstrated the presence of two tandemly-repeated lipoyl domains of approximately 125 amino acids in length, located at the Nterminus of the polypeptide. A short, highly conserved E3 binding sequence is found distal to the second lipoyl domain followed by the C-terminal intersubunit binding domain which also houses the acetyltransferase activity.…”