Subunit structure of the dihydrolipoyl transacylase component of branched-chain alpha-keto acid dehydrogenase complex from bovine liver. Characterization of the inner transacylase core.
Limited proteolysis has been used to probe the subunit structure (Mr = 52,000) of the dihydrolipoyl transacylase (E2) component of the branched-chain alpha-keto acid dehydrogenase complex from bovine liver. Digestion of the complex at 0 degrees C with a low concentration of trypsin produces an inner E2 core that retains the activity for the transacylation reaction and is completely dissociated from the decarboxylase (E1) component. The trypsinized E2 maintains the highly assembled structure and migrates faster than the native E2 in the Sepharose 4B column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the inner E2 core consists of two lipoate-free tryptic fragments, i.e. fragment A and fragment B with Mr = 26,000 and 22,000, respectively. Both fragments apparently fail to bind the E1 component. Fragment A is converted into fragment B by increasing trypsin concentrations. Fragment B is a stable limit polypeptide containing the intersubunit-binding sites for E2. The assemblage of fragment B confers the cubelike appearance of the inner E2 core in electron micrographs. Activity measurements indicate that the larger fragment A, but not fragment B, possesses transacylation activity. It is likely that a critical portion of the active site is present in the 4,000-dalton fragment that is lost during the conversion of fragment A to B.
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000217:Acyltransferases / Q000032:analysis*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D007658:Ketone Oxidoreductases / Q000032:analysis*; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000032:analysis*; D010446:Peptide Fragments / Q000032:analysis
D009097:Multienzyme Complexes; D010446:Peptide Fragments; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000217:Acyltransferases; C033588:dihydrolipoamide acyltransferase