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.