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A continuous fluorescent assay for protein prenyltransferases measuring diphosphate release
June E Pais1, Katherine E Bowers, Andrea K Stoddard
1Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Analytical Biochemistry
|September 7, 2005
Summary
A new assay measures prenyltransferase activity by tracking diphosphate release, enabling efficient screening for cancer drug targets. This method aids in identifying inhibitors for oncogenic Ras proteins involved in numerous human cancers.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Protein farnesyltransferase and geranylgeranyltransferase type I attach prenyl groups to proteins.
- These enzymes are crucial for oncogenic Ras proteins, which drive up to 30% of human cancers.
- Targeting prenyltransferases offers a promising strategy for cancer chemotherapy.
Purpose of the Study:
- To develop a coupled assay for measuring diphosphate (PPi) dissociation rates in prenyltransferase reactions.
- To validate this assay for both single and multiple turnover conditions.
- To enable high-throughput screening for prenyltransferase substrates and inhibitors.
Main Methods:
- A coupled enzymatic assay was designed to monitor PPi release.
- Inorganic pyrophosphatase rapidly cleaves PPi to phosphate (Pi).
- A coumarin-labeled phosphate-binding protein detects Pi, generating a fluorescence signal.
Main Results:
- The assay accurately measures the rate constant of PPi dissociation.
- This rate constant correlates with the prenylation rate constant.
- The assay is effective under both single and multiple turnover conditions.
- It is a nonradioactive method suitable for high-throughput screening.
Conclusions:
- A novel, nonradioactive coupled assay quantifies prenyltransferase activity.
- This assay facilitates the discovery of new chemotherapeutic agents targeting cancer-related prenyltransferases.
- The method is adaptable for high-throughput screening of potential drug candidates.