Protein farnesytransferase (PFTase) catalyzes the farnesylation of proteins with a carboxy-terminal tetrapeptide sequence denoted as a Ca1a2X box. To explore the specificity of this enzyme, an important therapeutic target, solid phase peptide synthesis in concert with a peptide inversion strategy were used to prepare two libraries, each containing 380 peptides. The libraries were screened using an alkyne-containing isoprenoid analogue followed by click chemistry with biotin azide and subsequent visualization with streptavidin-AP. Screening of the CVa2X and CCa2X libraries with R. norvegicus PFTase revealed reaction by many known recognition sequences as well as numerous unknown ones. Screening of the CVa2X library with alkyne-functionalized isoprenoid substrates showed that those prepared from C10 or C15 precursors gave similar results while the analogue synthesized from a C5 unit gave a different pattern of reactivity. Finally the substrate specificities of PFTases from three organisms (R. norvegicus,S. cerevisiae and C. albicans) were compared using CVa2X libraries. R. norvegicus PFTase was found to share more peptide substrates with S. cerevisiae PFTase than with C. albicans PFTase. In general, this method is a highly efficient strategy for rapidly probing the specificity of this important enzyme.

Rapid analysis of protein farnesyltransferase substrate specificity using peptide libraries

248th National Meeting (2014)

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