INVOLVEMENT OF THE ALPHA-SUBUNIT OF FARNESYL-PROTEIN TRANSFERASE IN SUBSTRATE RECOGNITION

Using photoaffinity labeling, we have identified a region in mammalian farnesyl-protein transferase (FPTase) involved in substrate recogniti on. The photolabel used (Compound 1) is a peptide containing the photo active amino acid p-benzoylphenylalanine (Bpa). Upon exposure to UV li ght, Compound 1 inhibits FPTase activity in a time- and concentration- dependent manner. Photoinhibition of FPTase activity by Compound 1 is prevented by adding I-I-Pas to the reaction mixture, indicating that l abeling is targeted to the enzyme active site. We used peptide mapping by HPLC, Edman sequencing, and matrix-assisted time-of-flight (MALDI- TOF) mass spectrometry to identify the site of interaction with radiol abeled Compound 1. These experiments indicate that a specific region o f the or subunit of the enzyme, Asp110-Arg112, is involved in substrat e binding and suggest that Glu111 is likely to be the residue covalent ly modified by the photoaffinity label. Sequence alignments between ye ast and mammalian FPTases reveal that Glu111 is conserved. The implica tions of this finding an discussed in light of previous mutagenesis st udies on FPTase.