Dynamics, Structure and Stability of α-Chymotrypsin in Aqueous Solution and in Reverse Micelles as Studied by Fluorescence Spectroscopy

Abstract

In the last few years different experimental approaches have been made to answer the question: how many water molecules does an enzyme require to display its enzymatic activity and to which extent has the protein globule to be hydrated to maintain the maximum conformational stability? The question under study requires to use a system in which the amount of water molecules can be very precisely controlled while the enzyme conformation-activity relationship can be followed by some very sensitive technique like fluorescence. Some amphiphilic surfactants like aerozol-OT (AOT) in organic solvents form thermodynamically stable, optically transparent reverse micelles in which aqueous solutions of an enzyme can be solubilized. The hydration degree, w o , defined as [H 2 O]/[AOT] is determined very precisely. As a model protein we have chosen α-chymotrypsin (CT) as one of the best studied enzymes. In addition to natural fluorescence of α-chymotrypsin a detailed fluorescence study of anthraniloyl-α-chymotrypsin (Ant-CT), in which a highly fluorescent anthraniloyl group was covalently attached to the active site, was carried out to investigate its physical properties in aqueous solution and in a restricted water system. Results presented in this study indicate a characteristic dependence for the stability and fluorescence properties of CT on the amount of water in the range of w o around 5. This feature is qualitatively discussed in terms of a “three phase model” of the state of water in reversed micelles (A. Goto et al. , Thermochim. Acta 163 (1990) 139). At very low w o the conformation of CT changes to very rigid in comparison to water solution. The four peaks in the fluorescence lifetime distribution in aqueous solution are converted into a single peak. The overall center of gravity of the tryptophan fluorescence spectrum of the enzyme at w o = 0.65 is blue shifted in comparison to the situation in water. In the absence of a hydration shell, the protein is essentially frozen and inactive. Small increases in water content transferred new water molecules to the negatively charged surfactants heads and the enzyme molecule becomes even more rigid than it is at w o = 0.65. The short correlation time peak of the anthraniloyl group, which represents the internal active site motion of CT shows an increase up to w o equal to about 5 where the enzyme displays a minimum of enzymatic activity. An increase in enzymatic activity within a relatively small w o range of 5 to 8 indicates a cooperative hydration-dependent activation of catalysis which accounts for cooperative binding of water molecules to the protein and covers a limited hydration range (< 1 g of water per gram of protein). Above w o = 10, however, the effect of hydration on the activation of catalysis 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 water in the system. In other words, bulk water is not necessary for activity. The results are consistent with the picture described by Rupley et al. (Adv. Chem., Ser. 80 (1980) 111).