Protein farnesyltransferase catalyzes the posttranslational farnesylation o
f several proteins involved in signal transduction, including Ras, and is a
target enzyme for antitumor therapies. Efficient product formation catalyz
ed by protein farnesyltransferase requires an enzyme-bound zinc cation and
high concentrations of magnesium ions. In this work, we have measured the p
H dependence of the chemical step of product formation, determined under si
ngle-turnover conditions, and have demonstrated that the prenylation rate c
onstant is enhanced by two deprotonations. Substitution of the active site
zinc by cadmium demonstrated that one of the ionizations reflects deprotona
tion of the metal-coordinated thiol of the peptide "CaaX" motif, pK(a1) = 6
.0. These data provide additional evidence for the direct involvement of a
metal-coordinated sulfur nucleophile in catalysis. The second ionization wa
s assigned to a hydroxyl on the pyrophosphate moiety of farnesyl pyrophosph
ate, pK(a2) = 7.4. Deprotonation of this group is important for binding of
magnesium. This second ionization is not observed for catalysis in the abse
nce of magnesium or when the substrate is farnesyl monophosphate. These dat
a indicate that the maximal rate constant for prenylation requires formatio
n of a zinc-coordinated thiolate nucleophile and enhancement of the electro
philic character at C1 of the farnesyl chain by magnesium ion coordination
of the pyrophosphate leaving group.