CRYOENZYMOLOGY - USE OF SUB-ZERO TEMPERATURES AND FLUID SOLUTIONS IN STUDY OF ENZYME MECHANISMS

Theoretical and experimental support for the use of sub-zero temperatures and fluid aqueous organic solvent systems in the study of enzyme mechanisms is presented. If the reaction between enzyme and substrate is initiated by mixing them at a suitably low temperature non-steady state, non-turnover conditions will result. Under such circumstances, the overall catalytic reaction will potentially appear as a series of reactions as the temperature is raised, each one corresponding to the transformation of one intermediate into a subsequent one. The technique is especially suited for the accumulation of high concentrations of intermediates which makes physical and chemical studies on their structure quite feasible. Recent investigations showed that enzyme-substrate intermediates can be trapped in the crystalline state and that X-ray crystallographic studies can be carried out on them at sub-zero temperatures. Rate reductions of the order of 1010 are possible using the combined effects of pH and temperature. Kinetic and thermodynamic information concerning intermediate transformations also may be obtained. Kinetic expressions for such systems are derived. The advantages and limitations of the method, including possible problems relating to the effects of solvent and temperature on the enzyme structure, experimental procedures and interpretation of results are considered.