Linked Life Data

8323281

Source:http://linkedlifedata.com/resource/pubmed/id/8323281

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1993-8-3
pubmed:abstractText
The protease encoded by the human immunodeficiency virus (HIV) is essential for the processing of viral polyproteins encoded by the gag and pol genes into mature viral proteins. The 99-residue protease from HIV-1 contains two cysteine residues (Cys-67 and Cys-95), both of which are usually conserved in viruses isolated from patients. Despite this conservation, neither residue is required for enzymatic activity. Certain site-specific cysteine mutants of HIV-1 protease are catalytically active, and the protease from HIV-2 lacks both cysteines. Copper is a potent inhibitor of HIV-1 protease, but not of mutants lacking cysteine (A. R. Karlström and R. L. Levine, 1991, Proc. Natl. Acad. Sci. USA 88, 5552-5556). The addition of copper to the protease at pH 5.5 induced aggregation of the protein, providing a possible basis for the inhibitory action of copper. However, addition of both copper and dithiothreitol still led to inhibition of activity but did not cause aggregation. These findings led to a study of the reactivity of the cysteine residues to 5,5'-dithiobis-(2-nitrobenzoic acid) (Ellman's reagent), a sulfhydryl compound which reacts with the ionized form of cysteine residues. At pH 6.2 in 6 M guanidine, no derivatization of cysteine residues occurred, consistent with the typical pKa of cysteine expected for the denatured protein. However, in the same buffer without guanidine, the native protease reacted rapidly with concomitant loss of proteolytic activity. Peptic mapping demonstrated that both Cys-67 and Cys-95 were derivatized. A catalytically active fusion protein of protease with protein A domains was then studied with the expectation that access to Cys-95 would be hindered. This was confirmed, with only Cys-67 reacting rapidly with Ellman's reagent. Enzymatic activity was again lost, indicating that derivatization of the surface-accessible Cys-67 was sufficient to inactivate the enzyme. The reactivity and accessibility of these residues suggest an interesting approach for the development of protease inhibitors which are not directed to the substrate-binding site.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
0003-9861
pubmed:author
pubmed:issnType
Print
pubmed:volume
304
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
163-9
pubmed:dateRevised
2001-11-13
pubmed:meshHeading
pubmed:year
1993
pubmed:articleTitle
Reactivity of cysteine residues in the protease from human immunodeficiency virus: identification of a surface-exposed region which affects enzyme function.
pubmed:affiliation
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
pubmed:publicationType
Journal Article