Regulation of HIV-1 protease activity through cysteine modification.
The homodimeric protease of the human immunodeficiency virus 1 contains two cysteine residues per monomer which are highly conserved among viral isolates. However, these cysteine residues are not essential for catalytic activity which raises the question of why they are conserved. We have found previously that these cysteine residues are unusually susceptible to oxidation by metal ions, and this results in inhibition of protease activity. Recombinant protease mutants (C67A, C95A, and the double mutant C67A,C95A) were prepared to assess the possible role of these cysteines in redox regulation of the enzyme. Mixed disulfides were formed between the cysteine residues of the enzymes and low molecular weight thiols. Enzyme activity was lost when a mixed disulfide was formed between 5,5'-dithiobis(2-nitrobenzoic acid) and cysteine 95, while the same mixed disulfide at cysteine 67 reduced activity by 50%. This effect was reversible as normal activity could be restored when the enzyme was treated with dithiothreitol. The cysteines could also be modified with the common cellular thiol glutathione. Modification with glutathione was verified by mass spectrometry of the protein peaks obtained from HPLC separation. Glutathiolation of cysteine 95 abolished activity whereas modification at cysteine 67 increased the k(cat) by more than 2-fold with no effect on K(m). In addition, glutathiolation at cysteine 67 markedly stabilized the enzyme activity presumably by reducing autoproteolysis. These results demonstrate one possible mechanism for regulation of the HIV-1 protease through cysteine modification and identify additional targets for affecting protease activity other than the active site.
Davis
D A
DA
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-0320, USA.
Dorsey
K
K
Wingfield
P T
PT
Stahl
S J
SJ
Kaufman
J
J
Fales
H M
HM
Levine
R L
RL
D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000235:genetics*; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005978:Glutathione / Q000378:metabolism; D016333:HIV Protease / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D010084:Oxidation-Reduction
D004228:Dithionitrobenzoic Acid; D016333:HIV Protease; D005978:Glutathione; D003545:Cysteine