Specific ion influences on self-association of pyruvate dehydrogenase kinase isoform 2 (PDHK2), binding of PDHK2 to the L2 lipoyl domain, and effects of the lipoyl group-binding site inhibitor, Nov3r

Association of the PDHK2 and GST-L2 (glutathione-S-transferase fused to the inner lipoyl domain (L2) of dihydrolipoyl acetyltransferase (E2)) dimers was enhanced by K⁺ with higher affinity K⁺ binding than occurs at the PDHK2 active site. Supporting a distinct K⁺ binding site, the NH₄⁺ ion did not effectively replace K⁺ in aiding GST-L2 binding. With 50 mM K⁺, P_i enhanced interference by ADP, ATP, or pyruvate of PDHK2 binding to GST-L2. The inclusion of P_i with ADP or ATP plus pyruvate greatly hindered PDHK2 binding to GST-L2 and promoted PDHK2 forming a tetramer. Reciprocally, GST-L2 interference with ATP/ADP binding also required elevated K⁺ and was increased by P_i. Potent inhibition by Nov3r of E2-activated PDHK2 activity (IC50 of ∼7.8 nM) required elevated K⁺ and P_i. Nov3r only modestly inhibited the low activity of PDHK2 without E2. By binding at the lipoyl group binding site, Nov3r prevented PDHK2 binding to E2 and GST-L2. Nov3r interfered with high-affinity binding of ADP and pyruvate via a P _i-dependent mechanism. Thus, GST-L2 binding to PDHK2 is supported by K⁺ binding at a site distinct from the active site. P_i makes major contributions to ligands interfering with PDHK2 binding to GST-L2, the conversion of PDHK2 dimer to a tetramer, and Nov3r (an acetyllipoate analog) interfering with binding of ADP and pyruvate. P_i is suggested to facilitate transmission within PDHK2 of the stimulatory signal of acetylation from the distal lipoyl-group binding site to the active site.