CONFORMATIONAL STABILITY AND CATALYTIC ACTIVITY OF HIV-1 PROTEASE AREBOTH ENHANCED AT HIGH-SALT CONCENTRATION

The activity of human immunodeficiency virus protease is markedly incr eased at elevated salt concentration. The structural basis of this eff ect 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 stabil ity (Delta G(0)) and the transition midpoint (D-1/2) between the folde d and unfolded states increase, indicating that the salt stabilizes th e enzyme structure. These equilibrium data are supported by kinetic st udies on the urea-mediated unfolding by measuring fluorescence change, red shifting in the maximum of the emission spectrum, and far-and nea r-UV CD. The salt effects observed in urea-mediated unfolding reaction s prevail upon heat denaturation. All these findings support the exist ence of a two-state equilibrium between the folded and unfolded protei ns. The pH dependence of fluorescence intensity indicated that the con formation of human immunodeficiency virus type 1 protease should chang e in the catalytically competent pH region. It is concluded that prefe rential hydration stabilizes the protease structure in the presence of salt, providing entropic contribution to enhance the catalytic activi ty.