Nematode pyruvate dehydrogenase kinases: role of the C-terminus in binding to the dihydrolipoyl transacetylase core of the pyruvate dehydrogenase complex

摘要

pPyruvate dehydrogenase kinases (PDKs) from the anaerobic parasitic nematode iAscaris suum/i and the free-living nematode iCaenorhabditis elegans/i were functionally expressed with hexahistidine tags at their N-termini and purified to apparent homogeneity. Both recombinant PDKs (rPDKs) were dimers, were not autophosphorylated and exhibited similar specific activities with the iA. suum/i pyruvate dehydrogenase (E1) as substrate. In addition, the activities of both PDKs were activated by incubation with PDK-depleted iA. suum/i muscle pyruvate dehydrogenase complex (PDC) and were stimulated by NADH and acetyl-CoA. However, the recombinant iA. suum/i PDK (rAPDK) required higher NADH/NADsup+/sup ratios for half-maximal stimulation than the recombinant iC. elegans/i PDK (rCPDK) or values reported for mammalian PDKs, as might be predicted by the more reduced microaerobic mitochondrial environment of the APDK. Limited tryptic digestion of both rPDKs yielded stable fragments truncated at the C-termini (trPDKs). The trPDKs retained their dimeric structure and exhibited substantial PDK activity with the iA. suum/i E1 as substrate, but PDK activity was not activated by incubation with PDK-depleted iA. suum/i PDC or stimulated by elevated NADH/NADsup+/sup or acetyl-CoA/CoA ratios. Direct-binding assays demonstrated that increasing amounts of rCPDK bound to the iA. suum/i PDK-depleted PDC. No additional rCPDK binding was observed at ratios greater than 20 mol of rCPDK/mol of PDC. In contrast, the truncated rCPDK (trCPDK) did not exhibit significant binding to the PDC. Similarly, a truncated form of rCPDK, rCPDKsub1–334/sub, generated by mutagenesis, exhibited properties similar to those observed for trCPDK. These results suggest that the C-terminus of the PDK is not required for subunit association of the homodimer or catalysis, but instead seems to be involved in the binding of the PDKs to the dihydrolipoyl transacetylase core of the complex./p