Reverse micelles as a water‐property‐control system to investigate the hydration/activity relationship of α‐chymotrypsin

Abstract

a-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, of approximately 5 (w, is defined as [H,O]/[AOT]). The results are consistent with heat-capacity measurements for proteins. At very low w, values, the conformation of a-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, = 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, = 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, = 5.1. A new increase of water content within a relatively small range, w, = 5-8, causes a dramatic increase in enzymic activity, reminiscent of a cooperative hydration dependence. In the range w, = 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, values followed by dilution to constant high w,, are indicative of inactive conformational substates of a-chymotrypsin. It is demonstrated that highly active enzyme conformations at very low w, values occur via an induced fit mechanism of substrate binding.Protein hydration plays a critical role in the catalytic activity and conformational stability of enzymes [l -31. From the analysis of hydrogen-isotope exchange data in proteins, it appears that the hydrogen-bonding network is a major factor in protein structure and function [4]. From the ability of the highly polar solvent methanol to desorb only 62% of water bound to enzymes [5], it has been suggested that at least two types of enzyme-bound water exist. The first type is structurally bound water. The second type is relatively easily removable water. It was shown that serine proteases contain an unusually large number of internal water molecules that are hydrogen bonded to the protein [6]. The positions of 7 out of 13 internally bound water molecules as found in ychymotrypsin are common sites for bound water in both achymotrypsin, diiso...

Ultrasonic Monitoring of Biocatalysis in Solutions and Complex Dispersions

“…According to Figure 11, the reaction in microemulsion proceeds significantly faster than in solution, which could be explained by a super-activity of the enzyme, often observed in microemulsions [70][71][72]. This conclusion is in agreement with the previous observations of the increase in the hydrolytic activity of the α-amylase against the oligomer substrates in a presence of surfactants [42].…”

Mechanism of protein extraction from the solid state by water-in-oil microemulsions

“…To achieve both of these objectives, a further understanding of the mechanism of protein solubilization into w/o-ME solution is required. Moreover, loss of enzyme activity frequently occurs during the incorporation procedure (Castro and Cabral, 1989;Dorovska-Taran et al, 1993;Han et al, 1990;Khmelnitsky et al, 1993). In general, the mechanism for protein solubilization and the structure of protein-containing w/o-MEs during and after the solubilization process are not fully understood.…”

Pressure-Induced Protein Unfolding in the Ternary System AOT−Octane−Water Is Different from that in Bulk Water

“…α-Chymotrypsin is a 25 kDa serine protease consisting of three chains connected by five disulfide bonds . Its catalytic activity in the AOT−octane−water system has been extensively characterized. ,, This has also included a study of the activity as a function of pressure. , However, to our knowledge, this is the first study of the conformational behavior of α-chymotrypsin in reverse micelles exposed to high pressure (up to 1.0 GPa).…”