Ursby Thomas, Weik Martin, Fioravanti Emanuela, Delarue Marc, Goeldner Maurice, Bourgeois Dominique
LCCP, UMR 9015, Institut de Biologie Structurale, 41 Avenue Jules Horowitz, 38027 Grenoble CEDEX 1, France.
Acta Crystallogr D Biol Crystallogr. 2002 Apr;58(Pt 4):607-14. doi: 10.1107/s0907444902002135. Epub 2002 Mar 22.
Caged compounds in combination with protein crystallography represent a valuable tool in studies of enzyme reaction intermediates. To date, photochemical triggering of reactions has been performed close to room temperature. Synchronous reaction initiation has only been achieved with enzymes of relatively slow turnover (<0.1 s(-1)) and caged compounds of high quantum yield. Here X-ray crystallography and microspectrophotometry were used to provide evidence that (nitrophenyl)ethyl (NPE) ester bonds can be photolyzed by UV light at cryotemperatures. NPE-caged ATP in flash-cooled crystals of Mycobacterium tuberculosis thymidylate kinase was photolyzed successfully at 100-150 K as assessed by the structural observation of ATP-dependent enzymatic conversion of TMP to TDP after temporarily warming the crystals to room temperature. A new method is proposed in which cryo-photolysis combined with temperature-controlled protein crystallography can be used to trap reaction intermediates even in some of the fastest enzymes and/or when only compounds of low quantum yield are available. Raising the temperature after cryophotolysis may allow a transition barrier to be passed and an intermediate to accumulate in the crystal. A comparable method has only been used so far with proteins displaying endogenous photosensitivity. The approach described here opens the way to studying the reaction mechanisms of a much larger number of crystalline enzymes. Furthermore, it is shown that X-ray-induced radiolysis of caged compounds occurs if high-intensity synchrotron beamlines are used. This caveat should be taken into account when deriving data-collection protocols. It could also be used potentially as a way to trigger reactions.
笼形化合物与蛋白质晶体学相结合，是研究酶反应中间体的一种有价值的工具。迄今为止，反应的光化学触发都是在接近室温的条件下进行的。仅在周转率相对较慢（<0.1 s⁻¹）的酶和高量子产率的笼形化合物中实现了同步反应引发。在这里，利用X射线晶体学和显微分光光度法提供证据表明，（硝基苯基）乙基（NPE）酯键在低温下可被紫外光光解。通过在将晶体临时升温至室温后对TMP向TDP的ATP依赖性酶促转化进行结构观察评估，结核分枝杆菌胸苷酸激酶的快速冷却晶体中的NPE-笼形ATP在100 - 150 K时成功光解。提出了一种新方法，其中低温光解与温度控制的蛋白质晶体学相结合，即使在一些最快的酶中，和/或当只有低量子产率的化合物可用时，也可用于捕获反应中间体。低温光解后升高温度可能使过渡屏障被跨越，中间体在晶体中积累。到目前为止，类似的方法仅用于显示内源性光敏性的蛋白质。这里描述的方法为研究大量结晶酶的反应机制开辟了道路。此外，研究表明，如果使用高强度同步加速器光束线，笼形化合物会发生X射线诱导的辐射分解。在推导数据收集方案时应考虑到这一注意事项。它也有可能被用作触发反应的一种方式。
