Conventional X-ray diffraction approaches to the study of enzyme mechanism: serine proteinases, aminoacyl-tRNA synthetases and xylose isomerase

Abstract

Techniques that have been used to study enzyme mechanism by conventional steady-state crystallographic techniques are reviewed. Substrates and substrate analogues can often be diffused into crystals, but occasionally co-crystallization is necessary. The poor solubility of substrates and inhibitors may pose a problem. Even if a substrate is present at adequate concentration, it may not be observed by X -ray diffraction. To observe a substrate, special measures may be needed to stop enzyme action, but sometimes this is not necessary because an equilibrium is established. Inhibitors may usefully model a particular reaction state, but one must always question whether the inhibitor provides a correct model. Stabilization of a transition state is often discussed, but rarely achieved. Where practicable, protein engineering can provide a powerful tool to test proposals about the catalytic mechanism. Molecular mechanics calculations can also be useful. These themes are developed in relation to enzymes studied in the authors’ laboratory. Many of the same problems are encountered in the application of time-resolved techniques to the study of enzyme mechanism.