H-Ras peptide and protein substrates bind protein farnesyltransferase as an ionized thiolate.

The zinc metalloenzyme protein farnesyltransferase (FTase) catalyzes the alkylation of a cysteine residue of protein substrates with a 15 carbon farnesyl group. We have developed fluorescence assays to directly measure the affinity of the enzyme for peptide and protein (Ras) substrates. A peptide corresponding to the carboxyl terminus of H-Ras binds to FTase in the microM range (KD = 4 microM) at physiological pH; however, the peptide affinity is enhanced approximately 70-fold in a ternary complex with an enzyme-bound farnesyl diphosphate (FPP) analogue, indicating that the two substrates bind synergistically. The pH dependence of substrate binding was also investigated, and two ionizations were observed: for the ternary complex, the pKa values are 8.1, reflecting ionization of the thiol of the free peptide, and 6.4. The pH dependence of the ligand-metal charge-transfer band in the optical absorption spectra of a Co2+-substituted FTase ternary complex suggests that a metal-coordinated thiol ionizes with a pKa of 6.3. These data indicate that metal coordination of the peptide sulfur with the zinc ion in FTase lowers the pKa of the thiol resulting in formation of a bound thiolate at physiological pH.