Duke E M, Hadfield A, Martin J L, Clifton I J, Hajdu J, Johnson L N, Reid G P, Trentham D R, Bruce I, Fleet G W
Laboratory of Molecular Biophysics, Oxford, UK.
Ciba Found Symp. 1991;161:75-86; discussion 86-90. doi: 10.1002/9780470514146.ch6.
Laue diffraction with high intensity, broad-spectrum synchrotron radiation sources allows three-dimensional data sets on protein crystals to be recorded in seconds or milliseconds and opens the way for time-resolved studies on dynamic events in crystals. This chapter briefly reviews the field and describes progress towards time-resolved studies with glycogen phosphorylase. Methods for the synchronization of the start of reaction with the start of data collection have been developed for the phosphorolytic reaction of glycogen phosphorylase. The compound 3,5-dinitrophenylphosphate is photolabile, yielding Pi and the by-product, 3,5-dinitrophenol, which is non-reactive with the enzyme. Spectroscopic studies show that the compound has good quantum yield and that photolysis is rapid (greater than 1000 s-1). Release of the dinitrophenylate anion, following a pulse of light from a xenon flash lamp, has been monitored with a diode array spectrophotometer specially adapted for measurements on crystals. In a laboratory X-ray experiment with crystals of glycogen phosphorylase b, release of Pi and formation of the enzyme-product complex have been demonstrated. The way is now open for Laue diffraction studies on the catalytic reaction in the crystal.
利用高强度、宽谱同步辐射源进行的劳厄衍射，能够在数秒或数毫秒内记录蛋白质晶体的三维数据集，为晶体中动态事件的时间分辨研究开辟了道路。本章简要回顾了该领域，并描述了糖原磷酸化酶时间分辨研究的进展。已开发出用于糖原磷酸化酶磷酸解反应的数据收集起始与反应起始同步的方法。化合物3,5-二硝基苯磷酸酯对光不稳定，会产生无机磷酸（Pi）和副产物3,5-二硝基苯酚，后者与该酶无反应性。光谱研究表明，该化合物具有良好的量子产率且光解迅速（大于1000 s-1）。用专门改装用于晶体测量的二极管阵列分光光度计监测了氙闪光灯脉冲照射后二硝基苯阴离子的释放。在糖原磷酸化酶b晶体的实验室X射线实验中，已证明了Pi的释放和酶-产物复合物的形成。现在开展晶体催化反应劳厄衍射研究的道路已经畅通。
