H-RAS PEPTIDE AND PROTEIN SUBSTRATES BIND PROTEIN FARNESYLTRANSFERASEAS AN IONIZED THIOLATE

The zinc metalloenzyme protein farnesyltransferase (FTase) catalyzes t he alkylation of a cysteine residue of protein substrates with a 15 ca rbon farnesyl group. We have developed fluorescence assays to directly measure the affinity of the enzyme for peptide and protein (Ras) subs trates. A peptide corresponding to the carboxyl terminus of H-Ras bind s to FTase in the mu M range (K-D = 4 mu M) at physiological pH; howev er, the peptide affinity is enhanced approximately 70-fold in a ternar y complex with an enzyme-bound farnesyl diphosphate (FPP) analogue, in dicating that the two substrates bind synergistically. The pH dependen ce of substrate binding was also investigated, and two ionizations wer e observed: for the ternary complex, the pK(a) values are 8.1, reflect ing ionization of the thiol of the free peptide, and 6.4. The pH depen dence of the ligand-metal charge-transfer band in the optical absorpti on spectra of a Co2+-substituted FTase ternary complex suggests that a metal-coordinated thiol ionizes with a pK(a) of 6.3. These data indic ate that metal coordination of the peptide sulfur with the zinc ion in FTase lowers the pK(a) of the thiol resulting in formation of a bound thiolate at physiological pH.