We have noted that the HIV-1 protease contains two cysteine residues, which are highly conserved among human immunodeficiency viral isolates. The cysteine residues are not required for catalytic activity, although metal-catalyzed oxidation of either cysteine causes loss of catalytic activity. These findings suggest an important role for the cysteines in viral production. A thorough understanding of the role of these cysteine residues could lead to alternative targets for inhibition of viral production. As reported previously, we found that the enzyme can be inhibited by metal complexes which do not require the cysteine residues for inhibition. The mechanism of inhibition by a BCDS-Cu (l) complex was studied and was found to be a competitive in nature. However, in vivo studies of the mechanism of inhibition of viral production by BCDS-Cu (l) demonstrated that it not due to inhibition of protease activity in infected cells. We now have evidence indicating that protease activity can be regulated through reversible modification of the cysteine residues. Using glutathione, mixed disulfides were formed at either cysteine residue and the effect on protease activity was evaluated. It was found that glutathiolation at Cys-67 increased enzyme activity almost 3 fold and protected the protein from self-degradation. In contrast, glutathiolation at Cys-95 abolished protease activity, and activity was restored upon removal of the glutathione with DTT. Restoration of protease activity could also be accomplished using a thioltransferase. These results indicate that disulfide formation at either the surface-exposed Cys-67 residue or the Cys-95 residue located near the dimer interface has significant but reversible effects on enzyme activity. This type of modification could take place in HIV-1 infected cells, especially those which are under oxidative stress.
