Conformational changes on substrate binding to methylmalonyl CoA mutase and new insights into the free radical mechanism
Conformational changes on substrate binding to methylmalonyl CoA mutase and new insights into the free radical mechanism
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DOI：
10.1016/s0969-2126(98)00073-2
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发表时间：
1998-06-15
期刊：
影响因子：
5.7
通讯作者：
Evans, PR
中科院分区：
文献类型：
--
作者：
Mancia, F;Evans, PR
背景：甲基丙二酰辅酶A通过自由基机制催化琥珀酰辅酶A和甲基丙二酰辅酶A的相互转化。该酶属于催化分子内重排反应的酶家族，其中相邻碳上的基团和氢原子交换。这些酶使用辅因子腺苷钴胺素（辅酶B-12），其断裂形成腺苷自由基，从而引发反应。确定无底物的甲基丙二酰辅酶A的结构，开始提供进一步深入了解自由基formation.Results的机制：我们在这里报告两个结构的甲基丙二酰辅酶A的谢氏丙酸杆菌。第一个结构是在2.5埃分辨率下与CoA的非生产性复合物中的酶。这种结构作为酶的无底物构象的模型，因为它非常类似于第二个差得多的2.7埃分辨率结构，该结构来自真正的无底物晶体。真正的无底物结构也显示腺苷基与钴原子结合。这种结构与以前报道的酶与底物类似物的复合物的比较表明，主要的构象变化发生在底物结合。该酶的底物结合位点位于（β α）（8）TIM桶结构域内。在不存在底物的情况下，该TIM-桶结构域分裂开，并且活性位点可接近溶剂。当基板结合，桶关闭了与基板沿着其轴和活性位点变得完全buried.Conclusions：关闭的活性位点腔后基板结合置换腺苷基团的辅因子从中央钴原子进入活性位点腔。这引发了引发重排反应的自由基的形成。TIM-桶结构域与所有其他已报道的结构域有很大的不同：在其未配体形式下，它被打破，暴露出填充中心的小亲水性侧链，典型的桶结构仅在底物结合时形成。
Background: Methylmalonyl CoA mutase catalyses the interconversion of succinyl CoA and methylmalonyl CoA via a free radical mechanism. The enzyme belongs to a family of enzymes that catalyse intramolecular rearrangement reactions in which a group and a hydrogen atom on adjacent carbons are exchanged. These enzymes use the cofactor adenosylcobalamin (coenzyme B-12) which breaks to form an adenosyl radical, thus initiating the reaction. Determination of the structure of substrate-free methylmalonyl CoA mutase was initiated to provide further insight into the mechanism of radical formation.Results: We report here two structures of methylmalonyl CoA mutase from Propionibacterium shermanii. The first structure is of the enzyme in a nonproductive complex with CoA at 2.5 Angstrom resolution. This structure serves as a model for the substrate-free conformation of the enzyme, as it is very similar to the second much poorer 2.7 Angstrom resolution structure derived from a truly substrate-free crystal. The true substrate-free structure also shows the adenosyl group bound to the cobalt atom. Comparison of this structure with that of the previously reported complex of the enzyme with a substrate analogue shows that major conformational changes occur upon substrate binding. The substrate-binding site of the enzyme is located within a (beta alpha)(8) TIM-barrel domain. In the absence of substrate, this TIM-barrel domain is split apart and the active site is accessible to solvent. When substrate binds, the barrel closes up with the substrate along its axis and the active site becomes completely buried.Conclusions: The closure of the active-site cavity upon substrate binding displaces the adenosyl group of the cofactor from the central cobalt atom into the active-site cavity. This triggers the formation of the free radical that initiates the rearrangement reaction. The TIM-barrel domain is substantially different from all others yet reported: in its unliganded form it is broken open, exposing the small hydrophilic sidechains which fill the centre, The typical barrel structure is only formed when substrate is bound.