Reverse micelles as a water-property-control system to investigate the hydration/activity relationship of alpha-chymotrypsin.

Abstract

alpha-Chymotrypsin, solubilized in hydrated reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in n-octane, was used as a model system for studying the involvement of different water structures (strongly bound water, disordered water, water clusters and bulk water) in the development of the catalytically active conformation of the enzyme. Results presented in this study indicate a characteristic dependence of the stability/activity profile on the water content of the reverse-micellar system for values of wo of approximately 5 (wo is defined as [H2O]/[AOT]). The results are consistent with heat-capacity measurements for proteins. At very low wo values, the conformation of alpha-chymotrypsin changes to a very rigid structure in comparison to the structure observed in water. This is demonstrated by the overall center of gravity of the tryptophan fluorescence spectrum of the enzyme at wo = 0.65, which is blue shifted in comparison to the spectrum in bulk water indicating that the enzyme is in an apolar environment. In the absence of a hydration shell, the protein is to a great extent frozen and inactive. A small increase in the level of enzyme hydration (up to wo = 2.3) causes an increase in the amount of strongly bound water associated with the enzyme and the enzyme displays a high catalytic activity. Upon further addition of water, a new unstable water structure with unfavourable enthalpy is developed and the enzyme activity declines, reaching a minimum at wo = 5.1. A new increase of water content within a relatively small range, wo = 5-8, causes a dramatic increase in enzymic activity, reminiscent of a cooperative hydration dependence. In the range wo = 10-29, the effect of hydration on the activity is complete which shows that the enzyme activity depends on the amount of water in contact with the enzyme and not on the total amount of bulk water in the system. The experimental results on enzyme incubation at different wo values followed by dilution to constant high wo, are indicative of inactive conformational substates of alpha-chymotrypsin. It is demonstrated that highly active enzyme conformations at very low, wo values occur via an induced fit mechanism of substrate binding.