Enzymology in supercooled water.

Abstract

RECENT years have witnessed an increasing interest in applications of low temperature techniques to studies of enzyme-catalysed reactions, the aims being the temporal resolution of the contributing reactions and the stabilisation of intermediate species. To overcome the low temperature limit set by the freezing point of the aqueous medium mixtures of water and various polar organic solvents have been used1–4. Although these aqueous mixtures affect the solvation interactions of the enzyme and its conformational stability5, they can be shown to influence neither substrate specificity, nor the reaction pathway. We have investigated the feasibility of supercooled water to achieve conditions closer to the physiological ones. To circumvent freezing by heterogeneous nucleation, the aqueous phase containing enzyme, substrate and buffers was emulsified in an oil which was supersaturated with a water insoluble surfactant6. We here present the results on bacterial cytochrome P450, a mono-oxygenase which has already been studied in mixed solvents1,2