THE HIV-1 PROTEASE AS ENZYME AND SUBSTRATE - MUTAGENESIS OF AUTOLYSISSITES AND GENERATION OF A STABLE MUTANT WITH RETAINED KINETIC-PROPERTIES

Site-directed mutagenesis of autolysis sites in the human immunodefici ency virus type 1 (HIV-1) protease was applied in an analysis of enzym e specificity; the protease served, therefore, as both enzyme and subs trate in this study. Inspection of natural substrates of all retrovira l proteases revealed the absence of beta-branched amino acids at the P -1 site and of Lys anywhere from P-2 through P-2'. Accordingly, severa l mutants of the HIV-1 protease were engineered in which these exclude d amino acids were substituted at their respective P positions at the three major sites of autolysis in the wild-type protease (Leu(5)-Trp(6 ), Leu(33)-Glu(34), and Leu(63)-Ile(64)), and the mutant enzymes were evaluated in terms of their resistance to autodegradation. All of the mutant HIV-1 proteases, expressed as inclusion bodies in Escherichia c oil, were enzymatically active after refolding, and all showed greatly diminished rates of cleavage at the altered autolysis sites. Some, ho wever, were not viable enzymatically because of poor physical characte ristics. This was the case for mutants having Lys replacements of Glu residues at P-2' and for another in which all three P-1 leucines were replaced by Ile. However, one of the mutant proteases, Q7K/L33I/L63I, was highly resistant to autolysis, while retaining the physical proper ties, specificity, and susceptibility to inhibition of the wild-type e nzyme. Q7K/L33I/L63I should find useful application as a stable surrog ate of the HIV-1 protease. Overall, our results can be interpreted rel ative to a model in which the active HIV-1 protease dimer is in equili brium with monomeric, disordered species which serve as the substrates for autolysis.