An enzyme-coupled continuous fluorescence assay for farnesyl diphosphate synthases

Jonathan K. Dozier, Mark D. Distefano

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

Farnesyl diphosphate synthase (FDPS) catalyzes the conversion of isopentenyl diphosphate and dimethylallyl diphosphate to farnesyl diphosphate, a crucial metabolic intermediate in the synthesis of cholesterol, ubiquinone, and prenylated proteins; consequently, much effort has gone into developing inhibitors that target FDPS. Currently most FDPS assays either use radiolabeled substrates and are discontinuous or monitor pyrophosphate release and not farnesyl diphosphate (FPP) creation. Here we report the development of a continuous coupled enzyme assay for FDPS activity that involves the subsequent incorporation of the FPP product of that reaction into a peptide via the action of protein farnesyltransferase (PFTase). By using a dansylated peptide whose fluorescence quantum yield increases upon farnesylation, the rate of FDPS-catalyzed FPP production can be measured. We show that this assay is more sensitive than existing coupled assays, that it can be used to conveniently monitor FDPS activity in a 96-well plate format, and that it can reproduce IC50 values for several previously reported FDPS inhibitors. This new method offers a simple, safe, and continuous method to assay FDPS activity that should greatly facilitate the screening of inhibitors of this important target.

Original languageEnglish (US)
Pages (from-to)158-163
Number of pages6
JournalAnalytical Biochemistry
Volume421
Issue number1
DOIs
StatePublished - Feb 1 2012

Bibliographical note

Funding Information:
This research was supported by the National Institutes of Health Grants GM58442 and GM084152 , as well as the National Institutes of Health Predoctoral Training Grant 5T32GM008700-13 . We thank Eric Oldfield (University of Illinois, Urbana-Champaign) for providing the FDPS inhibitors, and Claudia Schmidt-Dannert (University of Minnesota, Twin Cities) for providing the FDPS plasmid. We also thank Michael Hast and Lorena Beese (Duke University) for the creation of the yPFTase plasmid.

Keywords

  • Assay
  • Coupled assay
  • Diphosphate
  • Farnesyl diphosphate synthase
  • Fluorescence assay
  • Protein farnesyl transferase
  • Pyrophosphate

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