Mancia F, Evans P R
MRC Laboratory of Molecular Biology Hills Road, Cambridge, CB2 2QH, UK.
Structure. 1998 Jun 15;6(6):711-20. doi: 10.1016/s0969-2126(98)00073-2.
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 B12) 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.
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 A 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 A 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.
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.
甲基丙二酰辅酶A变位酶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。该酶属于一类催化分子内重排反应的酶家族，在这类反应中，相邻碳上的一个基团和一个氢原子会发生交换。这些酶利用辅因子腺苷钴胺素（辅酶B12），其分解形成腺苷自由基，从而引发反应。对无底物甲基丙二酰辅酶A变位酶结构的测定旨在进一步深入了解自由基形成的机制。
我们在此报告了来自谢氏丙酸杆菌的甲基丙二酰辅酶A变位酶的两种结构。第一种结构是该酶与辅酶A形成的非生产性复合物，分辨率为2.5埃。这种结构可作为该酶无底物构象的模型，因为它与从真正无底物晶体获得的分辨率低得多的第二种2.7埃结构非常相似。真正的无底物结构还显示腺苷基团与钴原子结合。将该结构与先前报道的该酶与底物类似物复合物的结构进行比较表明，底物结合时会发生主要的构象变化。该酶的底物结合位点位于一个（β-α）8 TIM桶状结构域内。在没有底物的情况下，这个TIM桶状结构域会分开，活性位点可被溶剂接触。当底物结合时，桶状结构会沿着其轴与底物一起闭合，活性位点完全被掩埋。
底物结合时活性位点腔的闭合将辅因子的腺苷基团从中心钴原子位移到活性位点腔内。这触发了引发重排反应的自由基的形成。TIM桶状结构域与迄今报道的所有其他结构域有很大不同：在其未结合配体的形式下它是打开的，暴露出填充中心的小亲水性侧链。典型的桶状结构仅在底物结合时形成。
