Molecular basis for specificities of reactivating factors for adenosylcobalamin‐dependent diol and glycerol dehydratases
Molecular basis for specificities of reactivating factors for adenosylcobalamin‐dependent diol and glycerol dehydratases
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DOI：
10.1111/j.1742-4658.2007.06074.x
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发表时间：
2007-11
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影响因子：
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通讯作者：
H. Kajiura;K. Mori;N. Shibata;T. Toraya
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作者：
H. Kajiura;K. Mori;N. Shibata;T. Toraya
腺苷钴胺素依赖性二醇和甘油脱氢酶是同功能酶，在催化过程中通过生理底物甘油进行基于机制的失活。失活的全酶被其自身的再活化因子再活化，所述再活化因子通过脱辅基酶的中间形成介导酶结合的受损辅因子与游离腺苷钴胺素的ATP依赖性交换。再活化分两步进行：（a）ADP依赖性钴胺素释放和（B）所得脱辅基酶-再活化因子复合物的ATP依赖性解离。纯化蛋白的体外实验表明，二醇脱氢酶再活化因子（DDR）交叉再活化失活的甘油脱氢酶，而甘油脱氢酶再活化因子（GDR）不交叉再活化失活的二醇脱氢酶。我们研究了它们的特异性在体外的分子基础，通过使用纯化的同源和非同源酶和再活化因子的制剂。DDR介导甘油脱氢酶结合的维生素B12与游离的腺嘌呤戊基维生素B12的交换，而GDR不能介导二醇脱氢酶结合的维生素B12与游离的腺嘌呤戊基维生素B12的交换。如通过变性PAGE所判断的，甘油脱氢酶-DDR复合物是交叉形成的，尽管没有形成二醇脱氢酶-GDR复合物。在酶-再活化因子复合物的ATP依赖性解离中，再活化因子没有特异性。因此，再活化因子的特异性很可能是由再活化因子与脱辅基酶形成复合物的能力决定的。基于酶和再活化因子的晶体结构的建模研究也表明了为什么DDR与甘油脱氢酶交叉形成复合物，以及为什么GDR不与二醇脱氢酶交叉形成复合物。
Adenosylcobalamin‐dependent diol and glycerol dehydratases are isofunctional enzymes and undergo mechanism‐based inactivation by a physiological substrate glycerol during catalysis. Inactivated holoenzymes are reactivated by their own reactivating factors that mediate the ATP‐dependent exchange of an enzyme‐bound, damaged cofactor for free adenosylcobalamin through intermediary formation of apoenzyme. The reactivation takes place in two steps: (a) ADP‐dependent cobalamin release and (b) ATP‐dependent dissociation of the resulting apoenzyme–reactivating factor complexes. The in vitro experiments with purified proteins indicated that diol dehydratase‐reactivating factor (DDR) cross‐reactivates the inactivated glycerol dehydratase, whereas glycerol dehydratase‐reactivating factor (GDR) did not cross‐reactivate the inactivated diol dehydratase. We investigated the molecular basis of their specificities in vitro by using purified preparations of cognate and noncognate enzymes and reactivating factors. DDR mediated the exchange of glycerol dehydratase‐bound cyanocobalamin for free adeninylpentylcobalamin, whereas GDR cannot mediate the exchange of diol dehydratase‐bound cyanocobalamin for free adeninylpentylcobalamin. As judged by denaturing PAGE, the glycerol dehydratase–DDR complex was cross‐formed, although the diol dehydratase–GDR complex was not formed. There were no specificities of reactivating factors in the ATP‐dependent dissociation of enzyme–reactivating factor complexes. Thus, it is very likely that the specificities of reactivating factors are determined by the capability of reactivating factors to form complexes with apoenzymes. A modeling study based on the crystal structures of enzymes and reactivating factors also suggested why DDR cross‐forms a complex with glycerol dehydratase, and why GDR does not cross‐form a complex with diol dehydratase.