Reactivity of cysteine-67 of the human immunodeficiency virus-1 protease: studies on a peptide spanning residues 59 to 75.
The protease encoded by the human immunodeficiency virus-1 (HIV-1) is essential for the processing of viral polyproteins into mature viral proteins. The 99-residue protease from HIV-1 contains two generally conserved cysteine residues, one of which (Cys-67) is located on the solvent-exposed surface. It was shown previously that Cys-67 of the native enzyme reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB, Ellman's reagent) at pH 6.2, causing a reversible inactivation of the protease. However, there was no reaction when the protein was denatured in 6 M guanidine hydrochloride, implying that the native conformation rendered Cys-67 more reactive. To investigate the structural basis of the lowered pKa in the native protein, we synthesized a 17-residue peptide matching the sequence of residues 59-75. The reactivity of this synthetic peptide with DTNB mimicked that of the protease, being more reactive in the absence of 6 M guanidine hydrochloride than in its presence. It was possible that His-69 or Lys-70 could facilitate ionization 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 guanidine 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 oxidation and disulfide formation. The resulting reversible inactivation of the protease may be important in the normal regulation of the viral life cycle, a suggestion supported by the strong conservation of the residues in this region.
D'Ettorre
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Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Levine
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D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D016333:HIV Protease / Q000737:chemistry*; D015497:HIV-1 / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry
D010455:Peptides; D004228:Dithionitrobenzoic Acid; D016333:HIV Protease; D003545:Cysteine