Farnesylation of nonpeptidic thiol compounds by protein farnesyltransferase

Protein farnesyltransferase catalyzes the modification of protein substrate s containing specific carboxyl-terminal Ca(1)a(2)X motifs with a 15-carbon farnesyl group. The thioether linkage is formed between the cysteine of the Ca(1)a(2)X motif and C1 of the farnesyl group. Protein substrate specifici ty is essential to the function of the enzyme and has been exploited to fin d enzyme-specific inhibitors for antitumor therapies. In this work, we inve stigate the thiol substrate specificity of protein farnesyltransferase by d emonstrating that a variety of nonpeptidic thiol compounds, including gluta thione and dithiothreitol, are substrates, However, the binding energy of t hese thiols is decreased 4-6 kcal/mol compared to a peptide derived from th e carboxyl terminus of H-Ras. Furthermore, for these thiol substrates, both the farnesylation rate constant and the apparent magnesium affinity decrea se significantly. Surprisingly, no correlation is observed between the pH-i ndependent log(k(max)) and the thiol pK(a); model nucleophilic reactions of thiols display a Bronsted correlation of approximately 0.4. These data dem onstrate that zinc-sulfur coordination is a primary criterion for classific ation as a FTase substrate, but other interactions between the peptide and the FTase isoprenoid complex provide significant enhancement of binding and catalysis. Finally, these results suggest that the mechanism of FTase prov ides in vivo selectivity for the farnesylation of protein substrates even i n the presence of high concentrations of intracellular thiols.