REACTIVITY OF CYSTEINE-67 OF THE HUMAN IMMUNODEFICIENCY VIRUS-1 PROTEASE - STUDIES ON A PEPTIDE SPANNING RESIDUE-59 TO RESIDUE-75

The protease encoded by the human immunodeficiency virus-1 (HIV-1) is essential for the processing of viral polyproteins into mature viral p roteins. The 99-residue protease from HIV-1 contains two generally con served cysteine residues, one of which (Cys-67) is located on the solv ent-exposed surface. It was shown previously that Cys-67 of the native enzyme reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB, Ellma n's reagent) at pH 6.2, causing a reversible inactivation of the prote ase. However, there was no reaction when the protein was denatured in 6 M guanidine hydrochloride, implying that the native conformation ren dered Cys-67 more reactive. To investigate the structural basis of the lowered pK(a) in the native protein, we synthesized a 17-residue pept ide matching the sequence of residues 59-75. The reactivity of this sy nthetic peptide with DTNB mimicked that of the protease, being more re active in the absence of 6 hi guanidine hydrochloride than in its pres ence. It was possible that His-69 or Lys-70 could facilitate ionizatio n of the SH group of Cys-67, which is required for reaction with DTNB. Apparently both residues are important, because increased reactivity of the native peptide was eliminated when either His-69 or Lys-70 were changed to Ala. Replacement of His-69 by Glu reversed the reactivity, so that the native peptide was less reactive than that denatured in g uanidine hydrochloride. Thus, the reactivity of Cys-67 is modulated by the charges on residues 69 and 70 in the protein. The presence of His -69 and Lys-70 renders the native protease especially susceptible to o xidation and disulfide formation. The resulting reversible inactivatio n of the protease may be important in the normal regulation of the vir al life cycle, a suggestion supported by the strong conservation of th e residues in this region.