REQUIREMENTS FOR THE ADAPTER PROTEIN ROLE OF DIHYDROLIPOYL ACETYLTRANSFERASE IN THE UP-REGULATED FUNCTION OF THE PYRUVATE-DEHYDROGENASE KINASE AND PYRUVATE-DEHYDROGENASE PHOSPHATASE

The dihydrolipoyl acetyltransferase (E2 component) is a 60-mer assembl ed via its COOH-terminal domain with exterior E1-binding domain and tw o lipoyl domains (L2 then L1) sequentially connected by mobile linker regions. E2 facilitates markedly enhanced function of the pyruvate deh ydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP), Human E2 structures were prepared with only one lipogl domain (L1 or L 2) or with alanines substituted at the sites of lipoylation (Lys-46 in L1 or Lys-173 in L2). The L2 domain and its lipoyl group were shown t o be essential for markedly enhanced PDP function and were required fo r greatly up-regulated PDR function. The complete absence of the L1 do main reduced the enhancements of both of these activities but not the maximal effector-stimulated PDK activity through acetylation of L2. Wi th nonlipoylated L2 present, lipoylated L1 supported a lesser enhancem ent in PDK function with significant stimulation upon acetylation of L 1. Prevention of L1 lipoylation in K46AE2 removed this competitive LI pole and enhanced La-facilitated PDK activity beyond that of native E2 when PDK activity was measured in the absence or in the presence of s timulatory effectors, Thus, the E2-L2 domain has a paramount role in f acilitating enhanced PDK and PDP function but inclusion of E2-L1 domai n, even in a noninteracting (nonlipoylated) form, contributes to the m arked elevation of these activities.