Additional binding sites for the pyruvate dehydrogenase kinase but not for protein X in the assembled core of the mammalian pyruvate dehydrogenase complex: binding region for the kinase.
A standard resolution of the bovine kidney pyruvate dehydrogenase complex yields a subcomplex composed of approximately 60 dihydrolipoyl transacetylase (E2) subunits, approximately 6 protein X subunits, and approximately 2 pyruvate dehydrogenase kinase heterodimers (KcKb). Using a preparation of resolved kinase in which Kc much greater than Kb, E2-X-KcKb subcomplex additionally bound at least 15 catalytic subunits of the kinase (Kc) and a much lower level of Kb. The binding of Kc to E2 greatly enhanced kinase activity even at high levels of bound kinase. Free protein X, functional in binding the E3 component, did not bind to E2-X-KcKb subcomplex. This pattern of binding Kc but not protein X was unchanged either with a preparation of E2 oligomer greatly reduced in protein X or with subcomplex from which the lipoyl domain of protein X was selectively removed. The bound inner domain of protein X associated with the latter subcomplex did not exchange with free protein X. These data support the conclusion that E2 subunits bind the Kc subunit of the kinase and suggest that the binding of the inner domain of protein X to the inner domain of the transacetylase occurs during the assembly of the oligomeric core. Selective release of a fragment of E2 subunits that contain the lipoyl domains (E2L fragment) releases the kinase (M. Rahmatullah et al., 1990, J. Biol. Chem. 265, 14,512-14,517). Sucrose gradient centrifugation yielded an E2L-kinase fraction with an increased ratio of the kinase to E2L fragment. A monoclonal antibody specific for E2L was attached to a gel matrix. Binding of E2L fragment also led to specific binding of the kinase. Extensive washing did not reduce the level of bound kinase. Thus, the kinase is tightly bound by the lipoyl domain region of E2.
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Department of Biochemistry, Kansas State University, Manhattan 66502.
Radke
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Ono
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Roche
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D000123:Acetyltransferases / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002417:Cattle; D002499:Centrifugation, Density Gradient; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D007668:Kidney / Q000201:enzymology; D046911:Macromolecular Substances; D003012:Microbial Collagenase / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010455:Peptides; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex; C489061:pyruvate dehydrogenase complex component X, Bos taurus; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D003012:Microbial Collagenase