Purpose <p>Following prenylation, some proteins undergo proteolytic removal of a C-terminal tripeptide in a reaction catalyzed by Ste24. This results in a protein terminating in a prenylcysteine residue that is subsequently methylated by a methyltransferase. In some cases, Ste24 also cleaves at an additional site upstream from the C-terminus. A central question in the field regards whether methylation is essential for subsequent upstream cleavage.</p> Methods <p>Three 33-residue peptides incorporating either a C-terminal methyl ester, a free acid or an amide were synthesized. Their preparation presented several synthetic challenges due to the presence of a C-terminal methyl ester, an epimerization-prone C-terminal cysteine and an acid-sensitive farnesyl group. The peptides were assayed for Ste24-catalyzed proteolysis using a fluorescence-based assay.</p> Results <p>The 33mer terminating in a C-terminal methyl ester was prepared using a side-chain anchoring strategy with minimal (&lt;5%) epimerization. The acid- and amide-terminating peptides were prepared using Wang resin and Rink MBHA amide resin, respectively, with the acid giving substantial (36%) epimerization. Collectively, the K_M and V_maxvalues for Ste24-catalyzed cleavage obtained using these three different peptides vary by a factor of 2 or less indicating that the identity of the C-terminal group does not significantly impact the rate of cleavage at the upstream site.</p> Conclusion <p>Side-chain anchoring provides a straight forward method for preparing peptides with C-terminal methyl esters with minimal epimerization. The demonstration that Ste24 cleaved the upstream site at rates that were not impacted by C-terminal structure suggests that carboxylmethylation is not a prerequisite for upstream cleavage by Ste24.</p>