Cryoenzymology.
A detailed understanding of the mechanism of enzyme action requires a correspondingly detailed knowledge of the structures of the intermediates and transition states on the catalytic pathway, as well as the kinetics and thermodynamics of their interconversion. Cryoenzymology, the use of subzero temperatures and fluid cryosolvents, has the potential to supply this type of information. In this article recent investigations illustrating the advantages of cryoenzymology are reviewed. The major advantage lies in the ability to accumulate and stabilize productive enzyme-substrate intermediates for sufficient lengths of time to allow the collection of high resolution structural data, e.g. by X-ray diffraction. In addition, intermediates which are not detectable under normal conditions, due to too short lifetimes and/or low concentrations, may be detected at low temperatures, in some cases through changes in the rate-limiting step. The method seems of rather general applicability, judging by the number of different types of enzymes, including oligomeric and membrane-associated ones, which have successfully been studied using cryoenzymology. In some cases, at least, good agreement between observations at subzero temperatures and normal conditions has been found, demonstrating the relevance of the technique. Potential limitations of the techniques, as well as questions regarding the effects on the protein structure, are also discussed.
D000818:Animals; D002736:Chloroplasts / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004355:Drug Stability; D003576:Electron Transport Complex IV / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D004798:Enzymes / Q000302:isolation & purification / Q000378:metabolism*; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D009994:Osmolar Concentration; D010447:Peptide Hydrolases / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D012697:Serine Endopeptidases; D013816:Thermodynamics
D004798:Enzymes; D003577:Cytochrome P-450 Enzyme System; D008156:Luciferases; D003576:Electron Transport Complex IV; D010450:Endopeptidases; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases