丙酮酸和2-氧戊二酸脱氢酶多酶复合物的单个重组成分(PDHc,OGDHc)的人和大肠杆菌(E.用优化的方案从大肠杆菌中表达和纯化大肠杆菌)来源。四种多酶复合物各自在最佳条件下以不同的化学计量比重构。从生理pH下复合物产生NADH的速率确定最高催化效率的结合化学计量。由于这些复合物中的一些被证明在通常伴有酸中毒的病理条件下具有“月光”活动,在pH6.3时也测定了活性。由于hOGDHc的E3成分产生活性氧(ROS)是病理相关的特征,通过乙酰化细胞色素c还原法在正向和反向催化方向上测量了具有最佳化学计量的复合物产生的超氧化物。各种已知的生理活动和ROS产生的影响因素,包括Ca(2+),ADP,脂化状态或pH,被调查了。人类复合物也与E3成分的最普遍的人类病理突变体重建,G194C和表征;先前报道具有G194C取代的分离的人E3具有增强的ROS产生能力。证明:i.PDHc,类似于OGDHc,能够产生ROS,并且该功能由大肠杆菌和人类复合物显示,Iftheparticularstandardsoftheparticularly,theparticularlytotheparticularlyofthebetweenthepurpose.当计算ROS生成的单位质量的共同E3组分时,与正向和反向反应中的hOGDHc相比,重构的hPDHc以明显更高的速率生成ROS,E1组分或E1-E2亚复合物仅在hOGDHc中产生显著量的ROS;iv.结合了hE3的G194C变体,这是一种致病突变的结果,转化为重组的hOGDHc和hPDHc确实导致两种复合物的活性降低,并且仅通过hOGDHc和仅在其逆反应中产生更高的ROS。
Individual recombinant components of pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes (PDHc, OGDHc) of human and Escherichia coli (E. coli) origin were expressed and purified from E. coli with optimized protocols. The four multienzyme complexes were each reconstituted under optimal conditions at different stoichiometric ratios. Binding stoichiometries for the highest catalytic efficiency were determined from the rate of NADH generation by the complexes at physiological pH. Since some of these complexes were shown to possess \'moonlighting\' activities under pathological conditions often accompanied by acidosis, activities were also determined at pH 6.3. As reactive oxygen species (ROS) generation by the E3 component of hOGDHc is a pathologically relevant feature, superoxide generation by the complexes with optimal stoichiometry was measured by the acetylated cytochrome c reduction method in both the forward and the reverse catalytic directions. Various known affectors of physiological activity and ROS production, including Ca(2+), ADP, lipoylation status or pH, were investigated. The human complexes were also reconstituted with the most prevalent human pathological mutant of the E3 component, G194C and characterized; isolated human E3 with the G194C substitution was previously reported to have an enhanced ROS generating capacity. It is demonstrated that: i. PDHc, similarly to OGDHc, is able to generate ROS and this feature is displayed by both the E. coli and human complexes, ii. Reconstituted hPDHc generates ROS at a significantly higher rate as compared to hOGDHc in both the forward and the reverse reactions when ROS generation is calculated for unit mass of their common E3 component, iii. The E1 component or E1-E2 subcomplex generates significant amount of ROS only in hOGDHc; iv. Incorporation of the G194C variant of hE3, the result of a disease-causing mutation, into reconstituted hOGDHc and hPDHc indeed leads to a decreased activity of both complexes and higher ROS generation by only hOGDHc and only in its reverse reaction.