New trends in baro-enzymology

Abstract

The recent interest on high pressure biotechnology (mainly in food industry) requires fundamental studies on the pressure behavior of biochemical constituants¹ In this laboratory, we use pressure as a thermodynamic parameter, such as temperature, to determine the energetic quantities of enzyme reactions². Two essential requirements for the study of the mechanism of enzyme action are that, first, a simple rate constant has to be measured (determination of a composite rate constant, such as kcat, can lead to ambiguous results) and, second, a maximum number of physico-chemical ways must be used to perturb the system under study. To assess this simple usually very rapid rate constant, cryoenzymology was used. It was then possible to probe, at constant pressure, the thermodynamics of the interconversion of two successive intermediates, thereby obtaining the classical ΔG‡ ΔS‡ and ΔH‡ parameters. Ify however, we can also vary another intensive parameter, namely the pressure, it is possible to determine the activation volume (ΔV‡) of the reaction. In addition to pressure and temperature, a third variable has to be considered also : the nature of the cryosolvent which allows experiments to be extended to subzero temperatures in the first place (with the medium being kept fluid) but which can also act as a perturbing agent thereby inducing controlled and reversible changes in equilibrium and me processes. The interdependence of the two variables, namely temperature and pressure, which is predicted by the general equation for the standard variation of free energy ΔG = f(T, P), is presented. This approach will be illustrated using different model reactions where data are analysed according to the classical transition state theory.