| Abstract: | For a full understanding of an enzyme reaction pathway, one must identify the reaction intermediates and obtain their structures and rates of interconversion. It is impossible to obtain all this information under normal conditions. An approach is to work suboptimally, in particular at subzero temperatures. This is cryoenzymology, an approach that implies both kinetic and structural measurements on enzyme systems below 0°C. To work below 0°C one must add an antifreeze so cryoenzymology means perturbation by two agents: temperature and antifreeze, usually an organic solvent. Certain precautions are needed with these agents, which we will discuss here. In particular, we discuss the importance of choosing the right solvent: this requires extensive exploratory studies but it is the key for the successful practice of cryoenzymology. Each system has its particularities and its own ‘good’ solvent. Cryoenzymology is not only a way of reducing reaction rates, it is also a way of perturbing one's system. Thus, it is a method that allows for the accumulation of intermediates that cannot be observed under normal conditions by slowing down their kinetics of formation, by changes in rate limiting steps or by shifts in equilibria. We illustrate the usefulness of cryoenzymology by myosin and actomyosin ATPases and by creatine, arginine and 3-phosphoglycerate kinases. We also discuss recent results obtained by X-ray crystallography. |
