DISSOCIATION AND REASSEMBLY OF THE DIHYDROLIPOYL TRANSACETYLASE COMPONENT OF THE BOVINE HEART PYRUVATE-DEHYDROGENASE COMPLEX

The catalytic activity and the state of aggregation of the dihydrolipoyl transacetylase-lipoamide dehydrogenase binding protein (E(2)-E(3)BP) subcomplex of the bovine heart pyruvate dehydrogenase multienzyme complex were investigated. Treatment of E(2)-E(3)BP with the chaotropic salts GndnCl or KSCN led to a rapid decrease in transacetylase activity which was accompanied by a loss of the native quaternary structure, as indicated by changes in the sedimentation properties of the E(2)-E(3)BP subcomplex. Reassembly or refolding of dissociated E(2)-E(3)BP was achieved for the GndnCl-treated subcomplex using a defined protocol. This reassembly procedure effectively excluded all E(3)BP from the reassembled oligomeric transacetylase. The reassembled oligomeric E(2), free of E(3)BP, was unable to reconstitute the overall activity of the complex following incubation with pyruvate dehydrogenase (E(3)) and lipoamide dehydrogenase (E(3)). in binding studies using radiolabeled components it was demonstrated that the reassembled transacetylase, while retaining its capacity for reductive acetylation and its ability to bind E(1), lost its ability to bind E(1). The evidence presented in this study indicates that the strong association of E(3)BP with E(2) facilitates the binding of E(3), the lipoamide dehydrogenase component, and therefore may have an important role in the assembly and ultimately the catalytic activity of the pyruvate dehydrogenase multienzyme complex. (C) 1995 Academic Press Inc.