Conversion of Tyr361 beta to Leu in mammalian protein farnesyltransferase impairs product release but not substrate recognition

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.