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    National Tsing Hua University Institutional Repository > 生命科學院  > 生命科學系 > 期刊論文 >  Identification of a Cysteine Residue Essential for Activity of Protein Farnesyltransferase: Cys299 is Exposed Only upon Removal of Zinc from the Enzyme.


    Please use this identifier to cite or link to this item: http://nthur.lib.nthu.edu.tw/dspace/handle/987654321/81423


    Title: Identification of a Cysteine Residue Essential for Activity of Protein Farnesyltransferase: Cys299 is Exposed Only upon Removal of Zinc from the Enzyme.
    Authors: Hua-Wen Fu;John F. Moomaw;Carolyn R. Moomaw;Patrick J. Casey
    教師: 傅化文
    Date: 1996
    Publisher: American Society for Biochemistry and Molecular Biology
    Relation: Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, Volume 271, Issue 45, NOV 1996, Pages 28541-28548
    Keywords: GERANYLGERANYLTRANSFERASE TYPE-I
    ALPHA-SUBUNIT
    METAL REQUIREMENTS
    KINETIC MECHANISM
    PEPTIDE-BINDING
    CDNA CLONING
    EXPRESSION
    RAT
    TRANSFERASE
    SYSTEM
    Abstract: Protein farnesyltransferase (FTase) is a zinc metalloenzyme that performs a post-translational modification on many proteins that is critical for their function. The importance of cysteine residues in FTase activity was investigated using cysteine-specific reagents. Zinc-depleted FTase (apo-FTase), but not the holoenzyme, was completely inactivated by treatment with N-ethylmaleimide (NEM). Similar effects were detected after treatment of the enzyme with iodoacetamide. The addition of zinc to apo-FTase protects it from inactivation by NEM. These findings indicated the presence of specific cysteine residue(s), potentially located at the zinc binding site, that are required for FTase activity. We performed a selective labeling strategy whereby the cysteine residues exposed upon removal of zinc from the enzyme were modified with [^3H]NEM. The enzyme so modified was digested with trypsin, and four labeled peptides were identified and sequenced, one peptide being the major site of labeling and the remaining three labeled to lesser extents. The major labeled peptide contained a radiolabeled cysteine residue, Cys^299, that is in the β subunit of FTase and is conserved in all known protein prenyltransferases. This cysteine residue was changed to both alanine and serine by site-directed mutagenesis, and the mutant proteins were produced in Escherichia coli and purified. While both mutant proteins retained the ability to bind farnesyl diphosphate, they were found to have lost essentially all catalytic activity and ability to bind zinc. These results indicate that the Cys^299 in the β subunit of FTase plays a critical role in catalysis by the enzyme and is likely to be one of the residues that directly coordinate the zinc atom in this enzyme.
    Relation Link: http://www.asbmb.org/
    URI: http://nthur.lib.nthu.edu.tw/dspace/handle/987654321/81423
    Appears in Collections:[分子與細胞生物研究所] 期刊論文
    [生命科學系] 期刊論文

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