REFOLDING AND RECONSTITUTION STUDIES ON THE TRANSACETYLASE PROTEIN-X (E2 X) SUBCOMPLEX OF THE MAMMALIAN PYRUVATE-DEHYDROGENASE COMPLEX - EVIDENCE FOR SPECIFIC BINDING OF THE DIHYDROLIPOAMIDE DEHYDROGENASE COMPONENT TO SITES ON REASSEMBLED E2/

Reconstitution studies have been conducted on the dihydrolipoamide ace tyltransferase-protein X core subcomplex of the mammalian pyruvate deh ydrogenase complex. GdnHCl-induced dissociation of this core is an ord ered cooperative event involving formation of specific lower-M-r inter mediates corresponding to dihydrolipoamide acetyltransferase trimers a nd monomers. Recovery profiles of the dihydrolipoamide acetyltransfera se-protein X core, unfolded in 6 M GdnHCl prior to the removal of dena turant by either (a) slow dialysis or (b) rapid dilution, demonstrated rapid initial reappearance of substantial levels of dihydrolipoamide acetyltransferase activity with complete recovery occurring in 4-6 h. Immunological analysis of reconstituted cores revealed reduced levels of protein X (approximately 30-35%) after slow dialysis and the total absence of this component following rapid dilution. The dihydrolipoami de acetyltransferase core, devoid of protein X, was unable to sustain overall complex activity when reconstituted with stoichiometric amount s of its companion pyruvate decarboxylase and dihydrolipoamide deydrog enase components, whereas the protein X-depleted core could sustain 30 -35% of control activity. Further reconstitution analyses of overall c omplex function with these two types of reassembled core structures in the presence of excess dihydrolipoamide dehydrogenase (100-fold) demo nstrated significant additional stimulation of pyruvate dehydrogenase complex activity (25-30%) which was dependent on the source of exogeno us dihydrolipoamide dehydrogenase. Thus, this constituent enzyme can i nteract directly with the dihydrolipoamide acetyltransferase oligomer with low affinity in addition to its normal high-affinity binding to t he protein X subunit, These results provide definitive in vitro eviden ce in support of recent clinical observations reporting residual pyruv ate dehydrogenase activity (10-20%) in cell lines derived from patient s lacking protein X.