Trifluoroethanol increases the stability of δ5-3-ketosteroid isomerase: 15N NMR relaxation studies

Abstract

In the equilibrium unfolding process of Δ5-3-ketosteroid isomerase from Pseudomonas testosteroni by urea, it was observed that the enzyme stability increases by 2.5 kcal/mol in the presence of 5% trifluoroethanol (TFE). To elucidate the increased enzyme stability by TFE, the backbone dynamics of Δ5-3-ketosteroid isomerase were studied in the presence and absence of 5% TFE by 15N NMR relaxation measurements, and the motional parameters (S2, τe, and Rex) were extracted from the relaxation data using the model-free formalism. The presence of 5% TFE causes little change or a slight increase in the order parameters (S2) for a number of residues, which are located mainly in the dimer interface region. However, the majority of the residues exhibit reduced order parameters in the presence of 5% TFE, indicating that high frequency (pico- to nanosecond) motions are generally enhanced by TFE. The results suggest that the entropy can be an important factor for the enzyme stability, and the increase in entropy by TFE is partially responsible for the increased stability of Δ5-3-ketosteroid isomerase.