Ramsay R R, Dreyer J L, Schloss J V, Jackson R H, Coles C J, Beinert H, Cleland W W, Singer T P
Biochemistry. 1981 Dec 22;20(26):7476-82. doi: 10.1021/bi00529a023.
It is known that aconitase from mammalian mitochondria is only partially active as isolated but may be activated by incubation with iron, ascorbate, and a thiol, or with dithionite. It has been suggested that the added Fe in the activation mixture is essential for activation and that it is incorporated in the enzyme [Villafranca, J. J., & Mildvan, A. S. (1971) J. Biol. Chem. 246, 772-779; Gawron, O., Waheed, A., Glaid, A. J., & Jaklitsch, A. (1974) Biochem. J. 139, 709-714]. However, it is shown in this paper that, when the enzyme has a full complement of 3Fe and 3S, full activation is reached coulometrically, without iron or other chemical reducing agents. It is clear, therefore, that the role of activators is to reduce the iron--sulfur cluster of the enzyme. The appearance of catalytic activity on reduction of the cluster shows a pronounced lag, as does the decay of activity after reoxidizing the cluster. This suggests that catalytic activity requires a conformational change in the protein which is initiated by reduction of the cluster and that, following reoxidation, activity disappears only after the inactive conformation is assumed. Citrate and the competitive inhibitor trans-aconitate are bound to a comparable extent to the active and inactive forms, but only the active form can bind 1-hydroxy-2-nitro-1,3-propanedicarboxylic acid, a transition-state analogue. This is interpreted to show that in the inactive state aconitase cannot enter the conformation it assumes in the transition state during catalysis.
已知哺乳动物线粒体中的乌头酸酶刚分离出来时只有部分活性，但与铁、抗坏血酸和一种硫醇一起孵育，或与连二亚硫酸盐一起孵育时可能会被激活。有人提出，激活混合物中添加的铁对激活至关重要，并且它会掺入酶中[Villafranca, J. J., & Mildvan, A. S. (1971) J. Biol. Chem. 246, 772 - 779; Gawron, O., Waheed, A., Glaid, A. J., & Jaklitsch, A. (1974) Biochem. J. 139, 709 - 714]。然而，本文表明，当酶具有完整的3个铁原子和3个硫原子时，通过库仑法可实现完全激活，无需铁或其他化学还原剂。因此，很明显激活剂的作用是还原酶的铁硫簇。簇还原时催化活性的出现有明显的延迟，簇再氧化后活性的衰减也是如此。这表明催化活性需要蛋白质发生构象变化，这种变化由簇的还原引发，并且再氧化后，只有在假定为无活性构象后活性才会消失。柠檬酸和竞争性抑制剂反乌头酸与活性形式和无活性形式的结合程度相当，但只有活性形式能结合1 - 羟基 - 2 - 硝基 - 1,3 - 丙二酸，一种过渡态类似物。这被解释为表明在无活性状态下乌头酸酶无法进入其在催化过程中过渡态所假定的构象。
