Conformational stability and catalytic activity of HIV-1 protease are both enhanced at high salt concentration.

The activity of human immunodeficiency virus protease is markedly increased at elevated salt concentration. The structural basis of this effect has been explored by several independent methods by using both the wild-type enzyme and its triple mutant (Q7K/L33I/L63I) (Mildner, A. M., Rothrock, D. J., Leone, J. W., Bannow, C. A., Lull, J. M., Reardon, I. M., Sarcich, J. L., Howe, W. J., Tomich, C.-S. C., Smith, C. W., Heinrikson, R. L., and Tomasselli, A. G. (1994) Biochemistry 33, 9405-9413), designed to better resist autolysis. Monitoring the intrinsic fluorescence of the two enzymes during urea-mediated denaturation has shown that at high NaCl concentration, both the conformational stability ( DeltaG0) and the transition midpoint (D1/2) between the folded and unfolded states increase, indicating that the salt stabilizes the enzyme structure. These equilibrium data are supported by kinetic studies on the urea-mediated unfolding by measuring fluorescence change, red shifting in the maximum of the emission spectrum, and far- and near-UV CD. The salt effects observed in urea-mediated unfolding reactions prevail upon heat denaturation. All these findings support the existence of a two-state equilibrium between the folded and unfolded proteins. The pH dependence of fluorescence intensity indicated that the conformation of human immunodeficiency virus type 1 protease should change in the catalytically competent pH region. It is concluded that preferential hydration stabilizes the protease structure in the presence of salt, providing entropic contribution to enhance the catalytic activity.