Photoaffinity labeling of Ras converting enzyme 1 (Rce1p) using a benzophenone-containing peptide substrate

Kelly Kyro, Surya P. Manandhar, Daniel Mullen, Walter K. Schmidt, Mark D. Distefano

Research output: Contribution to journalArticlepeer-review

7 Scopus citations


Isoprenylation is a post-translational modification that increases protein hydrophobicity and helps target certain proteins to membranes. Ras converting enzyme 1 (Rce1p) is an endoprotease that catalyzes the removal of a three residue fragment from the C-terminus of isoprenylated proteins. To obtain structural information about this membrane protein, photoaffinity labeling agents are being prepared and employed. Here, we describe the synthesis of a benzophenone-containing peptide substrate analogue for Rce1p. Using a continuous spectrofluorometric assay, this peptide was shown to be a substrate for Rce1p. Mass spectrometry was performed to confirm the site of cleavage and structure of the processed probe. Photolysis of the biotinylated compound in the presence of membranes containing Rce1p followed by streptavidin pull-down and Western blot analysis indicated that Rce1p had been labeled by the probe. Photolysis in the presence of both the biotinylated, benzophenone-containing probe and a farnesylated peptide competitor reduced the extent of labeling, suggesting that labeling is occurring in the active site.

Original languageEnglish (US)
Pages (from-to)5675-5684
Number of pages10
JournalBioorganic and Medicinal Chemistry
Issue number15
StatePublished - Aug 1 2010

Bibliographical note

Funding Information:
This research was supported by the National Institutes of Health Grants GM58442 (M.D.D.), and GM067092 (W.K.S.). Some equipment and materials were graciously supplied by Edgewood Chemical Biological Center. Additional thanks to Bruce Witthuhn at University of Minnesota Center for Mass Spectrometry and Proteomics.


  • Benzophenone
  • CaaX processing
  • Photoaffinity labeling
  • Prenyl protease
  • Protein isoprenylation
  • Proteolysis
  • Rce1p


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