Time-resolved macromolecular crystallography unifies structure determination with chemical kinetics, since the structures of transient states and chemical and kinetic mechanisms can be determined simultaneously from the same data. To start a reaction in an enzyme, typically, an initially inactive substrate present in the crystal is activated. This has particular disadvantages that are circumvented when active substrate is directly provided by diffusion. However, then it is prohibitive to use macroscopic crystals because diffusion times become too long. With small micro- and nanocrystals diffusion times are adequately short for most enzymes and the reaction can be swiftly initiated. We demonstrate here that a time-resolved crystallographic experiment becomes feasible by mixing substrate with enzyme nanocrystals which are subsequently injected into the X-ray beam of a pulsed X-ray source. 1. Enzymology The “unspent vital force” was the historical and metaphysical expression for a biological catalyst. It was eradicated in 1890 by an article worth almost 100 pages by O'Sullivan and Tompson [1], which probably denotes the beginning of what we know now as enzymology. The enzyme was originally thought to be the leaven in sour dough and then materialized as a chemical, a macromolecule, or something that can be manipulated, tested, and investigated. Similarities with chemical catalysts were discovered. This finally led to the nowadays well-established and important field of enzymology. In 1926 Sumner reported the purification and crystallization of the first enzyme, urease [2]. At that time, X-ray structure determination has been established starting in the first decade of the 20th century. Although the father of X-ray crystallography, Max von Laue, did not believe that it would be possible to determine atomic structures of enzymes with X-ray crystallography [3], the first structures of proteins were determined at the end of the 50s and the beginning of the 60s of the last century [4, 5]. Only then biologists learned to think in terms of 3D atomic structure of biological molecules. Structure-function relationships were discovered. Enzymes could be studied by directly seeing how, where, and in which orientation effectors were binding to the enzyme. Phenomena such as allosteric inhibition of enzyme activity first discovered by enzyme kineticists finally could be understood on the atomic length scale. This provided new venues, for example, to design new drugs to cure diseases. However, the atomic structures were still static. Enzymologists knew that catalysis must
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