A continuous fluorescent assay for protein prenyltransferases measuring diphosphate release

Protein farnesyltransferase and protein geranylgeranyltransferase type I catalyze the transfer of a 15- and a 20-carbon prenyl group, respectively, from a prenyl diphosphate to a cysteine residue at the carboxyl terminus of target proteins, with the concomitant release of diphosphate. Common substra...

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Bibliographic Details
Published in:Analytical biochemistry 2005-10, Vol.345 (2), p.302-311
Main Authors: Pais, June E., Bowers, Katherine E., Stoddard, Andrea K., Fierke, Carol A.
Format: Article
Language:English
Subjects:
Cloning, Molecular
Dimethylallyltranstransferase - analysis
Dimethylallyltranstransferase - metabolism
Diphosphate release
Diphosphates - analysis
Diphosphates - metabolism
DNA, Bacterial
Escherichia coli
Escherichia coli - genetics
Escherichia coli Proteins - metabolism
Fluorescence
Fluorescent Dyes - metabolism
Genes, Bacterial
Kinetics
Phosphate binding protein
Protein Binding
Protein farnesyltransferase
Protein geranylgeranyltransferase
Protein Prenylation
Tritium - metabolism
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Summary:Protein farnesyltransferase and protein geranylgeranyltransferase type I catalyze the transfer of a 15- and a 20-carbon prenyl group, respectively, from a prenyl diphosphate to a cysteine residue at the carboxyl terminus of target proteins, with the concomitant release of diphosphate. Common substrates include oncogenic Ras proteins, which are implicated in up to 30% of all human cancers, making prenyltransferases a viable target for chemotherapeutic drugs. A coupled assay has been developed to measure the rate constant of diphosphate (PP i) dissociation during the prenyltransferase reaction under both single and multiple turnover conditions. In this assay, the PP i group produced in the prenyltransferase reaction is rapidly cleaved by inorganic pyrophosphatase to form phosphate (P i), which is then bound by a coumarin-labeled phosphate binding protein from Escherichia coli, resulting in a fluorescence increase. The observed rate constant for PP i release is equal to the rate constant of prenylation of the peptide, as measured by other assays, so that this nonradioactive assay can be used to measure prenyltransferase activity under either single or multiple turnover conditions. This assay can be adapted for high-throughput screening for potential prenyltransferase substrates and inhibitors.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2005.07.040