Nematode pyruvate dehydrogenase kinases: role of the C-terminus in bindingto the dihydrolipoyl transacetylase core of the pyruvate dehydrogenase complex
W. Chen et al., Nematode pyruvate dehydrogenase kinases: role of the C-terminus in bindingto the dihydrolipoyl transacetylase core of the pyruvate dehydrogenase complex, BIOCHEM J, 339, 1999, pp. 103-109
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.