Subunit structure of dihydrolipoyl transacetylase component of pyruvate dehydrogenase complex from Escherichia coli.
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) and D (subunit binding domain). Release of fragments A from the complex does not significantly affect its sedimentation coefficient or its appearance in the electron microscope. Fragment A contains the lipoyl moieties ((3)H-labeled), is acidic with an apparent isoelectric point of about 4.0, has a M(r) of 31,600 as determined by sedimentation equilibrium analysis, and has a swollen or extended structure (f/f(o) = 1.78). Fragment A exhibits anomalous properties, probably due to its acidic nature. It is resistant to staining with Coomassie blue and it migrates on sodium dodecyl sulfate/polyacrylamide gels as if it had a M(r) of 46,000-48,000. Further tryptic digestion converts fragment A into a lipoyl-containing fragment of M(r) 20,000 (fragment B) and eventually into an apparently stable product of estimated M(r) about 10,000 (fragment C). Fragment D has a compact structure of M(r) about 29,600 as determined by sedimentation equilibrium analysis in 6 M guanidinium chloride, and it possesses the intersubunit binding sites of the transacetylase, the binding sites for pyruvate dehydrogenase and dihydrolipoyl dehydrogenase, and the catalytic site for transacetylation. The assemblage of fragments D is responsible for the cube-like appearance of the transacetylase in the electron microscope. High-resolution electron micrographs of the transacetylase show fiber-like extensions, apparently corresponding to tryptic fragment A, surrounding the cube-like core.
D000123:Acetyltransferases; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011768:Pyruvate Dehydrogenase Complex; D008063:Thioctic Acid / Q000031:analogs & derivatives
D046911:Macromolecular Substances; D010446:Peptide Fragments; D011768:Pyruvate Dehydrogenase Complex; D008063:Thioctic Acid; D000123:Acetyltransferases