Regulation of HIV-1 Protease Activity through Cysteine Modification
详细信息 查看全文
- 作者：David A. Davis ; Karen Dorsey ; Paul T. Wingfield ; Stephen J. Stahl ; Joshua Kaufman ; Henry M. Fales ; and Rodney L. Levine
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：February 20, 1996
- 年：1996
- 卷：35
- 期：7
- 页码：2482 - 2488
- 全文大小：343K
- 年卷期：v.35,no.7(February 20, 1996)
- ISSN：1520-4995
文摘
The homodimeric protease of the human immunodeficiency virus 1contains two cysteineresidues per monomer which are highly conserved among viral isolates.However, these cysteine residuesare not essential for catalytic activity which raises the question ofwhy they are conserved. We havefound previously that these cysteine residues are unusually susceptibleto oxidation by metal ions, andthis results in inhibition of protease activity. Recombinantprotease mutants (C67A, C95A, and the doublemutant C67A,C95A) were prepared to assess the possible role ofthese cysteines in redox regulation ofthe enzyme. Mixed disulfides were formed between the cysteineresidues of the enzymes and low molecularweight thiols. Enzyme activity was lost when a mixed disulfide wasformed between 5,5'-dithiobis(2-nitrobenzoic acid) and cysteine 95, while the same mixed disulfide atcysteine 67 reduced activity by50%. This effect was reversible as normal activity could berestored when the enzyme was treated withdithiothreitol. The cysteines could also be modified with thecommon cellular thiol glutathione.Modification with glutathione was verified by mass spectrometry ofthe protein peaks obtained fromHPLC separation. Glutathiolation of cysteine 95 abolished activitywhereas modification at cysteine 67increased the kcat by more than 2-fold with noeffect on Km. In addition, glutathiolationat cysteine 67markedly stabilized the enzyme activity presumably by reducingautoproteolysis. These results demonstrateone possible mechanism for regulation of the HIV-1 protease throughcysteine modification and identifyadditional targets for affecting protease activity other than theactive site.