Lauble H, Stout C D
Institut für Organische Chemie, Universität Stuttgart, Germany.
Proteins. 1995 May;22(1):1-11. doi: 10.1002/prot.340220102.
Crystal structures of mitochondrial aconitase with alpha-methylisocitrate and with sulfate bound have been solved and refined at 2.0 A resolution with R factors of 18.2 and 16.8%, respectively. The steric factors and conformational effects observed in both new structures support the proposed mechanism for the overall reaction catalyzed by aconitase. The alternate substrate alpha-methylisocitrate is derived from alpha-methyl-cis-aconitate during crystallization and is observed to bind in the active site in a manner very similar to that observed for isocitrate. The methyl group is accommodated by favorable contact with Ile-425. However, the other potential hydration product of alpha-methyl-cis-aconitate, alpha-methylcitrate, cannot be accommodated in the active site due to steric conflict of the methyl group with Asp-165. The results are consistent with the requirement that cis-aconitate must bind in two ways, in the citrate mode and in the isocitrate mode. Crystals of aconitase with sulfate bound are isomorphous to those with isocitrate bound. However, the structure displays significant conformational changes, providing a model for the substrate-free state of enzyme. Three water molecules bind in place of the C alpha- and C beta-hydroxyl and carboxyl groups of isocitrate, while sulfate binds in place of the C gamma-carboxyl group. Side chains of Ser-642 and Arg-447 in the active site rotate to pair with other side chains in the absence of substrate. The new conformation of Arg-447 triggers a concerted set of shifts which transmits conformational change to the surface of the protein, 30 A from the active site. In the absence of substrate, a chain segment containing the [4Fe-4S] ligand Cys-358 also shifts, resulting in the net translation and reorientation of the Fe-S cluster.
已解析并精修了与α-甲基异柠檬酸结合以及与硫酸根结合的线粒体乌头酸酶的晶体结构，分辨率为2.0 Å，R因子分别为18.2%和16.8%。在这两种新结构中观察到的空间因素和构象效应支持了乌头酸酶催化整体反应的 proposed 机制。替代底物α-甲基异柠檬酸在结晶过程中由α-甲基顺乌头酸衍生而来，并且观察到它以与异柠檬酸非常相似的方式结合在活性位点。甲基通过与Ile-425的良好接触得以容纳。然而，α-甲基顺乌头酸的另一种潜在水合产物α-甲基柠檬酸，由于甲基与Asp-165的空间冲突而无法容纳在活性位点。结果与顺乌头酸必须以两种方式结合的要求一致，即柠檬酸模式和异柠檬酸模式。与硫酸根结合的乌头酸酶晶体与与异柠檬酸结合的晶体同晶型。然而，该结构显示出显著的构象变化，为酶的无底物状态提供了一个模型。三个水分子取代异柠檬酸的Cα-和Cβ-羟基以及羧基而结合，而硫酸根取代Cγ-羧基。在没有底物的情况下，活性位点中的Ser-642和Arg-447的侧链旋转以与其他侧链配对。Arg-447的新构象引发了一系列协同的位移，将构象变化传递到距活性位点30 Å的蛋白质表面。在没有底物的情况下，包含[4Fe-4S]配体Cys-358的链段也会发生位移，导致Fe-S簇的净平移和重新定向。
