Assays for S-adenosylmethionine (AdoMet/SAM)-dependent methyltransferases

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Methyltransferases play crucial roles in biological processes by modifying small molecules and proteins.
• Accurate measurement of methyltransferase activity is essential for understanding these processes and for drug development.
• Existing assays can be limited by product feedback inhibition from S-adenosyl-L-homocysteine (AdoHcy).

Purpose of the Study:

• To develop and validate novel, high-throughput methods for measuring methyltransferase activity.
• To overcome limitations of existing assays, particularly product feedback inhibition.
• To provide robust and cost-effective tools for biochemical and enzymatic research.

Main Methods:

• Development of an enzyme-coupled continuous spectrophotometric assay monitoring the hydrolysis of S-adenosyl-L-homocysteine (AdoHcy) via adenine intermediates.
• Implementation of a discontinuous assay measuring radiolabel incorporation into the methyl acceptor.
• Utilized recombinant AdoHcy nucleosidase and adenine deaminase enzymes to process AdoHcy and enable continuous monitoring.

Main Results:

• Both developed assays accurately quantify S-adenosyl-L-methionine (AdoMet or SAM)-dependent methyltransferase activity.
• The assays effectively destroy AdoHcy, alleviating feedback inhibition and allowing for more reliable measurements.
• The spectrophotometric assay allows continuous monitoring via absorbance changes at 265 nm.

Conclusions:

• The new spectrophotometric and radiolabel assays are inexpensive, robust, and suitable for high-throughput screening.
• These methods provide valuable tools for studying methyltransferase function in various biological contexts.
• Overcoming AdoHcy feedback inhibition enhances the utility of these assays for biochemical research and drug discovery.