CRYOENZYMOLOGY - HOW TO PRACTICE KINETIC AND STRUCTURAL STUDIES

For a full understanding of an enzyme reaction pathway, one must ident ify the reaction intermediates and obtain their stuctures and rates of interconversion. It is impossible to obtain all this information unde r normal conditions. An approach is to work suboptimally, in particula r at subzero temperatures. This is cryoenzymology, an approach that im plies both kinetic and structural measurements on enzyme systems below 0 degrees C. To work below 0 degrees C one must add an antifreeze so cryoenzymology means perturbation by two agents: temperature and antif reeze, usually an organic solvent. Certain precautions are needed with these agents, which we will discuss here. In particular, we discuss t he importance of choosing the right solvent: this requires extensive e xploratory studies but it is the key for the successful practice of cr yoenzymology. Each system has its particularities and its own 'good' s olvent. Cryoenzymology is not only a way of reducing reaction rates, i t 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 u nder normal conditions by slowing down their kinetics of formation, by changes in rate limiting steps or by shifts in equilibria. We illustr ate the usefulness of cryoenzymology by myosin and actomyosin ATPases and by creatine, arginine and 3-phosphoglycerate kinases. We also disc uss recent results obtained by X-ray crystallography.