Ra. Spence et al., Conversion of Tyr361 beta to Leu in mammalian protein farnesyltransferase impairs product release but not substrate recognition, BIOCHEM, 39(45), 2000, pp. 13651-13659
Protein farnesyltransferase catalyzes the lipid modification of protein sub
strates containing Met, Ser, Gin, or Ala at their C-terminus. A closely rel
ated enzyme, protein geranylgeranyltransferase type I, carries out a simila
r modification of protein substrates containing a C-terminal Leu residue. A
nalysis of a mutant of protein farnesyltransferase containing a Tyr-to-Leu
substitution at position 361 in the beta subunit led to the conclusion that
the side chain of this Tyr residue played a major role in recognition of t
he protein substrates, However, no interactions have been observed between
this Tyr residue and peptide substrates in the crystal structures of protei
n farnesyltransferase. In an attempt to reconcile these apparently conflict
ing data, a thorough kinetic characterization of the Y361L variant of mamma
lian protein farnesyltransferase was performed. Direct binding measurements
for the Y361L variant yielded peptide substrate binding that was actually
some 40-fold tighter than that with the wild-type enzyme. In contrast, bind
ing of the peptide substrate for protein geranylgeranyltransferase type I w
as very weak. The basis for the discrepancy was uncovered in a pre-steady-s
tate kinetic analysis, which revealed that the Y361L variant catalyzed farn
esylation of a normal peptide substrate at a rate similar to that of the wi
ld-type enzyme in a single turnover, but that subsequent turnover was preve
nted. These and additional studies revealed that the Y361L variant does not
"switch" protein substrate specificity as concluded from steady-state para
meters; rather, this variant exhibits severely impaired product dissociatio
n with its normal substrate, a situation resulting in a greatly compromised
steady-state activity.