Combined pressure and cosolvent effects on enzyme activity – a high-pressure stopped-flow kinetic study on α-chymotrypsin

Abstract

We investigated the combined effects of cosolvents and pressure on the hydrolysis of a model peptide catalysed by α-chymotrypsin. The enzymatic activity was measured in the pressure range from 0.1 to 200 MPa using a high-pressure stopped-flow systems with 10 ms time resolution. A kosmotropic (trimethalymine-N-oxide, TMAO) and chaotropic (urea) cosolvent and mixtures thereof were used as cosolvents. High pressure enhances the hydrolysis rate as a consequence of a negative activation volume, ΔV^#, which, depending on the cosolvent system, amounts to −2 to −4 mL mol⁻¹. A more negative activation volume can be explained by a smaller compression of the ES complex relative to the transition state. Kinetic constants, such as k_cat and the Michaelis constant K_M, were determined for all solution conditions as a function of pressure. With increasing pressure, k_cat increases by about 35% and its pressure dependence by a factor of 1.9 upon addition of 2 M urea, whereas 1 M TMAO has no significant effect on k_cat and its pressure dependence. Similarly, K_M increases upon addition of urea 6-fold. Addition of TMAO compensates the urea-effect on k_cat and K_M to some extent. The maximum rate of the enzymatic reaction increases with increasing pressure in all solutions except in the TMAO : urea 1 : 2 mixture, where, remarkably, pressure is found to have no effect on the rate of the enzymatic reaction anymore. Our data clearly show that compatible solutes can easily override deleterious effects of harsh environmental conditions, such as high hydrostatic pressures in the 100 MPa range, which is the maximum pressure encountered in the deep biosphere on Earth.