SUBUNIT INTERACTIONS IN THE MAMMALIAN ALPHA-KETOGLUTARATE DEHYDROGENASE COMPLEX - EVIDENCE FOR DIRECT ASSOCIATION OF THE ALPHA-KETOGLUTARATE DEHYDROGENASE AND DIHYDROLIPOAMIDE DEHYDROGENASE COMPONENTS

Selective tryptic proteolysis of the mammalian alpha-ketoglutarate deh ydrogenase complex (OGDC) leads to its rapid inactivation as a result of a single cleavage within the N-terminal region of its alpha-ketoglu tarate dehydrogenase (E1) component, which promotes the dissociation o f the dihydrolipoamide dehydrogenase (E3) enzyme smd also a fully acti ve E1' fragment. Similarities between the N-terminal region of E1 and the dihydrolipoamide acetyltransferase (E2) and E2-binding components (E3BP) of the pyruvate dehydrogenase complex are highlighted by the sp ecific cross-reactivities of subunit-specific antisera. Analysis of th e pattern of release of E1 and E1' polypeptides from the OGDC during t ryptic inactivation suggests that both polypeptide chains of individua l E1 homodimers must be cleaved to permit the dissociation of the E1 a nd E3 components. A new protocol has been devised that promotes E1 dis sociation from the oligomeric dihydrolipoamide succinyltransferase (E2 ) core in an active state. Significant Bevels of overall OGDC reconsti tution could also be achieved by re-mixing the constituent enzymes in stoichiometric amounts. Moreover, a high affinity interaction has been demonstrated between the homodimeric E1 and E3 components, which form a stable subcomplex comprising single copies of these two enzymes.