Chen W, Komuniecki P R, Komuniecki R
Department of Biology, The University of Toledo, Toledo, OH 43606-3390, USA.
Biochem J. 1999 Apr 1;339 ( Pt 1)(Pt 1):103-9.
Pyruvate dehydrogenase kinases (PDKs) from the anaerobic parasitic nematode Ascaris suum and the free-living nematode Caenorhabditis elegans 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 A. suum pyruvate dehydrogenase (E1) 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 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 A. suum E1 as substrate, but PDK activity was not activated by incubation with PDK-depleted A. suum PDC or stimulated by elevated NADH/NAD+ or acetyl-CoA/CoA ratios. Direct-binding assays demonstrated that increasing amounts of rCPDK bound to the A. suum 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, rCPDK1-334, 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 catalysis, but instead seems to be involved in the binding of the PDKs to the dihydrolipoyl transacetylase core of the complex.
来自厌氧寄生线虫猪蛔虫和自由生活线虫秀丽隐杆线虫的丙酮酸脱氢酶激酶（PDKs）在其N端带有六聚组氨酸标签进行功能表达，并纯化至表观均一。两种重组PDKs（rPDKs）均为二聚体，不会自磷酸化，并且以猪蛔虫丙酮酸脱氢酶（E1）为底物时表现出相似的比活性。此外，两种PDKs的活性通过与去除PDK的猪蛔虫肌肉丙酮酸脱氢酶复合物（PDC）孵育而被激活，并受到NADH和乙酰辅酶A的刺激。然而，重组猪蛔虫PDK（rAPDK）达到半数最大刺激所需的NADH/NAD⁺比值高于重组秀丽隐杆线虫PDK（rCPDK）或哺乳动物PDKs报道的值，这正如猪蛔虫PDK所处的微需氧线粒体环境更为还原所预测的那样。对两种rPDKs进行有限的胰蛋白酶消化产生了在C端截短的稳定片段（trPDKs）。trPDKs保留了其二聚体结构，并以猪蛔虫E1为底物表现出大量的PDK活性，但PDK活性不会通过与去除PDK的猪蛔虫PDC孵育而被激活，也不会受到升高的NADH/NAD⁺或乙酰辅酶A/辅酶A比值的刺激。直接结合试验表明，越来越多的rCPDK与去除猪蛔虫PDK的PDC结合。当rCPDK与PDC的摩尔比大于20时，未观察到额外的rCPDK结合。相比之下，截短的rCPDK（trCPDK）与PDC没有显著结合。同样，通过诱变产生的rCPDK截短形式rCPDK1 - 334表现出与trCPDK相似的特性。这些结果表明，PDK的C端对于同型二聚体的亚基缔合或催化不是必需的，而是似乎参与了PDKs与复合物的二氢硫辛酰胺转乙酰酶核心的结合。
