Open AccessReverse micelles as a water‐property‐control system to investigate the hydration/activity relationship of α‐chymotrypsin
Author(s) -
DOROVSKATARAN V.,
VEEGER C.,
VISSER A. J. W. G.
Publication year - 1993
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1993.tb18460.x
Subject(s) - chemistry , micelle , chymotrypsin , bound water , water activity , enzyme , enzyme assay , enthalpy , crystallography , water content , chemical engineering , organic chemistry , thermodynamics , trypsin , molecule , aqueous solution , physics , geotechnical engineering , engineering
α‐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 w o of approximately 5 ( w o is defined as [H 2 O]/[AOT]). The results are consistent with heat‐capacity measurements for proteins. At very low w o values, the conformation of α‐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 w o = 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 w o = 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 w o = 5.1. A new increase of water content within a relatively small range, w o = 5–8, causes a dramatic increase in enzymic activity, reminiscent of a cooperative hydration dependence. In the range w o = 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 w o values followed by dilution to constant high w o , are indicative of inactive conformational substates of α‐chymotrypsin. It is demonstrated that highly active enzyme conformations at very low w o values occur via an induced fit mechanism of substrate binding.