Mechanism of Aconitase Action
Mechanism of Aconitase Action
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乌头酸酶作用机制
DOI：
10.1016/s0021-9258(18)96082-5
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
E. O'Connell
中科院分区：
文献类型：
--
作者：
I. A. Rose;E. O'Connell
提出以下方案来表示氚在乌头酸酶（乌头酸水合酶）反应中的路径。[see提供了支持该方案的五个实验。1. 3-T-异柠檬酸盐在早期转化为柠檬酸盐，得到T-柠檬酸盐，没有氚的损失。当通过用异柠檬酸脱氢酶捕获异柠檬酸而完成2-T-柠檬酸向异柠檬酸的转化时，用谷氨酸脱氢酶形成α-酮戊二酸，在谷氨酸中发现的氚量与基于从柠檬酸形成异柠檬酸和顺乌头酸的相对初始速率的预测一致.在没有高浓度顺乌头酸盐存在下，氘从柠檬酸盐向异柠檬酸盐的转移主要是分子内的.在高浓度的顺乌头酸盐中，一些通常存在于水中的2-T-柠檬酸的氚被转移到异柠檬酸盐中.这种氚的守恒是由于分子间的转移，因为在高顺乌头酸盐存在下，氚化的2-甲基羟基酸生成氚化的异柠檬酸盐，这些数据支持了碱催化质子从羟基酸底物中提取的机理。在酶上如此形成的共轭酸基团解离缓慢，或者在顺乌头酸解离之前转移到结合的顺乌头酸上，或者，如果顺乌头酸被释放，共轭酸基团解离以再生酶碱。在羟基酸的相互转化中，很少或没有18 O标记的羟基基团转移。
The following scheme is proposed to represent the path of tritium in the aconitase (aconitate hydratase) reaction.[see PDF for equation]Five experiments that support this scheme are presented.1. The conversion of 3-T-isocitrate to citrate at early times gives T-citrate with no loss of tritium.2. When the conversion of 2-T-citrate to isocitrate is brought to completion by trapping the isocitrate with isocitrate dehydrogenase, and the α-ketoglutarate formed with glutamate dehydrogenase, the amount of tritium found in the glutamate agrees with the prediction based on the relative initial rates of formation of isocitrate andcis-aconitate from citrate.3. The transfer of deuterium from citrate to isocitrate in the absence of high concentrations ofcis-aconitate is shown to be largely intramolecular.4. At high concentrations ofcis-aconitate some of the tritium of 2-T-citrate that is normally found in the water is diverted to isocitrate.5. This conservation of tritium is due to an intermolecular transfer since in the presence of highcis-aconitate the tritiated 2-methyl hydroxy acids give rise to tritiated isocitrate.These data support a mechanism of base-catalyzed proton abstraction from hydroxy acid substrate. The conjugate acid group so formed on the enzyme is slow to dissociate and either is transferred to boundcis-aconitate before the latter dissociates, or, if thecis-aconitate is released, the conjugate acid group dissociates to regenerate the enzyme base.In the interconversion of the hydroxy acids there is little or no transfer of18O-labeled hydroxyl group.