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

Abstract

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) The pyruvate dehydrogenase complex from Escherichia coli is organized about a core, consisting of dihydrolipoyl transacetylase, to which pyruvate dehydrogenase and dihydrolipoyl dehydrogenase are joined by noncovalent bonds (1). The appearance of the transacetylase in the electron microscope is that of a cube, and its design appears to be based on octahedral (432) symmetry (2). The transacetylase consists of 24 apparently identical polypeptide chains, and each transacetylase chain apparently contains two (3-5) or possibly three (6) covalently bound lipoyl moieties. Lipoic acid is bound in amide linkage to the E-amino group of a lysyl residue (7). The lipoyl moiety is thought to rotate among the catalytic centers of the complex (8). This communication reports the results of studies of limited tryptic digestion of the E. coli dihydrolipoyl transacetylase in which novel aspects of its subunit structure were revealed. It appears that the native transacetylase subunit contains a compact domain of Mr -29,600 that possesses all the intersubunit binding sites as well as the catalytic site. To this domain is attached a flexible extension of Mr -31,600 containing the lipoyl moieties. MATERIALS AND METHODSDL-[2-3H]Lipoic acid (30,000-44,000 cpm/,ug) was synthesized by metalation of lipoic acid at C-2 (9) followed by treatment with 3H20 (J. R. Butler and L. J. Reed, unpublished data). N-Ethyl[2,3-14C]maleimide was purchased from Amersham/ Searle. L-(1-Tosylamido-2-phenyl)ethyl chloromethyl ketone-treated trypsin was obtained from Worthington, and soybean trypsin inhibitor (type 1-S) was from Sigma.Pyruvate dehydrogenase complex containing [2-3H]lipoic acid covalently bound to the dihydrolipoyl transacetylase component was isolated from E. coli (Crookes strain) grown in the presence of 102-125 ,ug of DL-[2-3H]lipoic acid per liter of culture medium as described (8, 10 Sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electrophoresis was performed in 7.5% acrylamide gels by the procedure of Weber and Osborn (12). The samples were heated at 1000 C for 3-5 min. MrS were estimated from these gels by use of suitable marker proteins (10). For radioactivity measurement the gels were sliced into 1-or 2-mm sections, and the slices were dissolved in 30% hydrogen peroxide. Alternatively, N-N'-diallyl tartardiamide (Eastman) was used instead of methylene bisacrylamide as the crosslinking agent, and the gel slices were dissolved in 2% periodic acid (13). Radioactivity was determined in ACS cocktail (Amersham/Searle) in a Beckman LS-230 scintillation counter.Mrs were determined by sedimentation equilibrium with the meniscus depletion technique (14). The interference photographs were measured with an automatic scanner (15), and the data were evaluated by a computer program (16). Mrs were obtained from the in...

Purification and characterization of acetoin:2,6-dichlorophenolindophenol oxidoreductase, dihydrolipoamide dehydrogenase, and dihydrolipoamide acetyltransferase of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system

“…The subunits of dihydrolipoamide acyltransferases from other organisms typically of one to three lipoyl-binding domain and one catalytic domain, which are connected through proteasesensitive hinge regions (5,9,20,28,40). Upon limited proteolysis under nondenaturing conditions, the hinge regions are cut and usually two fragments with similar molecular masses, representing the lipoyl-binding domains and the catalytic domain, were obtained.…”

“…Dihydrolipoamide acyltransferases (E2 components) of 2-oxo acid dehydrogenase complexes from different sources are very sensitive to proteolytic cleavage under nondenaturing conditions (5,9,20,28,40). After limited proteolysis, a domain comprising the lipoyl moieties (designated the lipoylbinding domain) and a domain containing the acetyltransferase activity (designated the catalytic or subunit-binding domain) are usually obtained.…”

Antibodies against an inter‐domain segment of polypeptide chain inhibit active‐site coupling in the pyruvate dehydrogenase multienzyme complex

“…On the basis of their susceptibility to limited proteolysis [1,3,31] and their identification as the origin of many of the unexpectedly sharp signals detected by 1H-NMR spectroscopy [10][11][12][13], the long (alanine + proline)-rich segments of the E. coli E2p chain are thought to be exposed to solvent and conformationally flexible. Perhaps it is not so surprising, therefore, that peptide PEP3 is strongly immunogenic and that the anti-PEP3 Fab fragments can bind effectively to the PDH complex.…”

Conformational mobility of polypeptide chains in the 2‐oxo acid dehydrogenase complexes from ox heart revealed by proton NMR spectroscopy

“…Studies of the pyruvate dehydrogenase complexes from E. coli [8][9][10], from B. stearothermophilus [ 11 ] and from ox kidney [ 12] and ox heart [ 13] by limited proteolysis and electron microscopy suggested that lipoic acid-containing regions of the E2 chains protrude from the E2 core between the E1 and E3 subunits. An extraordinary degree of conformational mobility of large segments of the E2 chains encompassing the lipoyl-lysine residues was demonstrated by proton NMR spectroscopy of the E. coli complex [ 14].…”