Harmych Sally, Arnette Robin, Komuniecki Richard
Department of Biological Sciences, The University of Toledo, Toledo, OH 43606-3390, USA.
Mol Biochem Parasitol. 2002 Nov-Dec;125(1-2):135-46. doi: 10.1016/s0166-6851(02)00221-9.
The pyruvate dehydrogenase complex (PDC) plays changing roles during the aerobic-anaerobic transition in the life cycle of the parasitic nematode, Ascaris suum. However, the dihydrolipoyl dehydrogenase (E3) subunit appears to be identical in all stages, despite the fact that the PDC is less sensitive to NADH inhibition in anaerobic muscle. Therefore, we have cloned cDNAs encoding E3 and a novel anaerobic-specific E3-binding protein (E3BP) that lacks the terminal lipoyl domain found in E3BPs from yeast and mammals, and functionally expressed E3 and E3 mutants designed to have decreased dimer stability on the assumption that the binding of E3 to an anaerobic-specific E3BP might stabilize the E3 dimer interface and decrease E3 sensitivity to NADH inhibition. As predicted, the mutants exhibited decreased thermal stability, increased sensitivity to NADH and the binding of E3(Y18F) to the E3-depleted core of the pig heart PDC increased E3 activity and decreased E3 sensitivity to NADH inhibition. However, although the free A. suum E3 was less sensitive to NADH inhibition than the pig heart E3, both E3s were significantly more sensitive to NADH inhibition when assayed with dihydrolipoamide than their corresponding PDCs assayed with pyruvate. More importantly, the binding of rE3 to its core complex had little effect on its apparent K(m) for NAD(+), K(i) for NADH inhibition, or the NADH/NAD(+) ratio yielding 50% inhibition. These data suggest that although binding to the core stabilizes the E3 dimer interface, it does not play a significant role in reducing the sensitivity of the A. suum PDC to NADH inhibition during anaerobiosis.
丙酮酸脱氢酶复合体（PDC）在寄生线虫猪蛔虫的生命周期的有氧 - 无氧转变过程中发挥着不断变化的作用。然而，尽管在无氧肌肉中PDC对NADH抑制的敏感性较低，但二氢硫辛酰胺脱氢酶（E3）亚基在所有阶段似乎都是相同的。因此，我们克隆了编码E3和一种新型厌氧特异性E3结合蛋白（E3BP）的cDNA，该蛋白缺乏酵母和哺乳动物的E3BPs中发现的末端硫辛酰结构域，并在功能上表达了E3和设计为具有降低二聚体稳定性的E3突变体，假设E3与厌氧特异性E3BP的结合可能会稳定E3二聚体界面并降低E3对NADH抑制的敏感性。正如预测的那样，这些突变体表现出热稳定性降低、对NADH的敏感性增加，并且E3（Y18F）与猪心PDC的E3缺失核心的结合增加了E3活性并降低了E3对NADH抑制的敏感性。然而，尽管游离的猪蛔虫E3对NADH抑制的敏感性低于猪心E3，但当用二氢硫辛酰胺测定时，两种E3对NADH抑制的敏感性都明显高于用丙酮酸测定的相应PDC。更重要的是，重组E3与其核心复合体的结合对其NAD⁺的表观Kₘ、NADH抑制的Kᵢ或产生50%抑制的NADH/NAD⁺比值几乎没有影响。这些数据表明，尽管与核心的结合稳定了E3二聚体界面，但在厌氧过程中，它在降低猪蛔虫PDC对NADH抑制的敏感性方面并没有发挥重要作用。
