Reference: Rozema DB and Poulter CD (1999) Yeast protein farnesyltransferase. pKas of peptide substrates bound as zinc thiolates. Biochemistry 38(40):13138-46

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Abstract


Protein farnesyltransferase (PFTase) is a zinc metalloenzyme that catalyzes the posttranslational alkylation of the cysteine in C-terminal -Ca(1)a(2)X sequences by a 15-carbon farnesyl residue, where C is cysteine, a(1) and a(2) are normally aliphatic amino acids, and X is an amino acid that specifies selectivity for the farnesyl moiety. Formation of a Zn(2+) thiolate in the PFTase. peptide complex was detected by the appearance of an absorbance at 236 nm (epsilon = 15 000 M(-1) cm(-1)), which was dependent on the concentration of peptide, in a UV difference spectrum in a solution of PFTase and the peptide substrate RTRCVIA. We developed a fluorescence anisotropy binding assay to measure the dissociation constants as a function of pH for peptide analogues by appending a 2',7'-difluorofluorescein to their N-terminus. The electron-withdrawing fluorine atoms allowed us to measure peptide binding down to pH 5.5 without having to correct for the changes in fluorescence intensity that accompany protonation of the fluorophore. Measurements of the pK(a)s for thiol groups in free and bound peptide indicate that peptide binding is accompanied by formation of a zinc thiolate and that binding to PFTase lowers the pK of the peptide thiol by 3 units. In similar studies with the betaY310F mutant, the pK(a) of the thiol moiety was lowered by 2 units upon binding, indicating that the hydroxyl group in the conserved tyrosine helps stabilize the bound thiolate.

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Journal Article | Research Support, U.S. Gov't, Non-P.H.S. | Research Support, U.S. Gov't, P.H.S.
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Rozema DB, Poulter CD
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