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

Pyruvate dehydrogenase kinases (PDKs) from the anaerobic parasitic nematode Ascaris suum and the free-living nematode Caenorhabditis elegans were func tionally expressed with hexahistidine tags at their N-termini and purified to apparent homogeneity. Both recombinant PDKs (rPDKs) were dimers, were no t autophosphorylated and exhibited similar specific activities with the A. suum pyruvate dehydrogenase (El) as substrate. In addition, the activities of both PDKs were activated by incubation with PDK-depleted A. suum muscle pyruvate dehydrogenase complex (PDC) and were stimulated by NADH and acetyl -CoA. However, the recombinant A. suum PDK (rAPDK) required higher NADH/NAD (+) ratios for half-maximal stimulation than the recombinant C. elegans PDK (rCPDK) or values reported for mammalian PDKs, as might be predicted by th e 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 exhibite d substantial PDK activity with the A. suum El as substrate, but PDK activi ty was not activated by incubation with PDK-depleted A, suum PDC or stimula ted by elevated NADH/NAD(+) or acetyl-CoA/CoA ratios. Direct-binding assays demonstrated that increasing amounts of rCPDK bound to the A. suum PDK-dep leted 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 n ot exhibit significant binding to the PDC. Similarly, a truncated form of r CPDK, rCPDK(1-334), 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 catalys is, but instead seems to be involved in the binding of the PDKs to the dihy drolipoyl transacetylase core of the complex.