Yeast protein farnesyltransferase. pK(a)s of peptide substrates bound as zinc thiolates

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 specifi es selectivity for the farnesyl moiety. Formation of a Zn2+ thiolate in the PFTase peptide complex was detected by the appearance of an absorbance at 236 nm (is an element of = 15 000 M(-1)cm(-1)), which was dependent on the concentration of peptide, in a UV difference spectrum in a solution of PFTa se and the peptide substrate RTRCVIA. We developed a fluorescence anisotrop y binding assay to measure the dissociation constants as a function of pH f or peptide analogues by appending a 2',7'-difluorofluorescein to their N-te rminus. The electron-withdrawing fluorine atoms allowed us to measure pepti de binding down to pH 5.5 without having to correct for the changes in fluo rescence intensity that accompany protonation of the fluorophore. Measureme nts 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 bin ding to PFTase lowers the pK of the peptide thiol by 3 units. In similar st udies with the beta Y310F mutant, the pK(a) of the thiol moiety was lowered by 2 units upon binding, indicating that the hydroxyl group in the conserv ed tyrosine helps stabilize the bound thiolate.