Art is long and time is fleeting: the current problems and future prospects for time-resolved enzyme crystallography

I offer comments on the challenges and problems of the future based on the papers in this volume. First, the requirement of the Laue technique for a very well-ordered crystal is a major obstacle to many studies. Efforts to ease this problem are needed. Secondly, the fundamental issues in time-resolved crystallography are now chemical rather than crystallographic. Methods for the rapid initiation of many reactions must be developed. Thirdly, it is imperative that the kinetics of the process in question be studied in the crystal before any diffraction experiments are done. We need better ways to make those solid state kinetic measurements. Fourthly, we should make use of combined methods, such as cryoenzymology plus Laue diffraction or site-directed mutagenesis plus Laue diffraction, to bring many processes into the time regime in which we currently can work. Fifthly, we have to be able to deconvolute diffraction data that come from a mixture of two or three discrete species. Finally, no matter how powerful our synchrotrons get, it seems to me that some of the most important events in any enzymatic reaction are not going to be accessible: consider the formation and decomposition of a transition state as an example. I close by discussing the role of computational biochemistry in filling in those frames of our enzymatic movie that we cannot observe directly by time-resolved X-ray crystallography.

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