Weston S A, Tucker A D, Thatcher D R, Derbyshire D J, Pauptit R A
Protein Structure Laboratory, Zeneca Pharmaceuticals, Macclesfield, Cheshire, United Kingdom.
J Mol Biol. 1994 Dec 9;244(4):410-22. doi: 10.1006/jmbi.1994.1739.
Ricin is a potent plant toxin which acts by removing a specific adenine residue from the ribosome. The X-ray crystal structure of a new, tetragonal crystal form of the recombinant ricin A-chain diffracting to 1.8 A resolution has been determined via molecular replacement methods and refined to a crystallographic R-factor of 18.6%. The higher resolution electron density allowed improvements to be made upon previously published models, resulting in an increase in the assigned secondary structure of the protein. The enzyme adopts the same global conformation in this crystal form with differences in detail due only partly to crystal packing. The active site superimposes closely with those of previously published models but the locations of the active-site water molecules differ in this structure. To address the current mechanistic model, an additional two structures are presented: recombinant ricin A-chain complexed with the substrate analogue formycin monophosphate as well as with adenosine monophosphate, which is cleaved by the crystalline enzyme. The formycin monophosphate displaces a putative catalytic water molecule. This supports the notion that the analogue does not bind in a transition state conformation and that contacts from other elements of the 28 S RNA natural substrate are required to achieve full reactivity. The structure of the adenosine monophosphate complex suggests a mechanism for the release of the adenine product via of the side-chain Tyr80. The structures suggest that Glu177 is better positioned for the activation of the catalytic water molecule than Arg180.
蓖麻毒素是一种强效植物毒素，其作用方式是从核糖体上移除特定的腺嘌呤残基。通过分子置换方法确定了重组蓖麻毒素A链一种新的四方晶型的X射线晶体结构，该结构的衍射分辨率为1.8埃，并精修至晶体学R因子为18.6%。更高分辨率的电子密度使得能够对先前发表的模型进行改进，从而增加了蛋白质中指定的二级结构。在这种晶体形式中，该酶采用相同的整体构象，细节上的差异仅部分归因于晶体堆积。活性位点与先前发表的模型紧密重叠，但在此结构中活性位点水分子的位置有所不同。为了探讨当前的作用机制模型，还展示了另外两种结构：重组蓖麻毒素A链与底物类似物单磷酸间型霉素以及与被晶体酶切割的单磷酸腺苷形成的复合物。单磷酸间型霉素取代了一个假定的催化水分子。这支持了这样一种观点，即该类似物不是以过渡态构象结合，并且需要28 S RNA天然底物的其他元件的接触才能实现完全反应性。单磷酸腺苷复合物的结构表明了通过侧链Tyr80释放腺嘌呤产物的机制。这些结构表明，与Arg180相比，Glu177更有利于催化水分子的活化。
