A continuous kinetic assay for protein and DNA methyltransferase enzymatic activities

Shai Duchin, Zlata Vershinin, Dan Levy, Amir Aharoni

Methyltransferases (MTs) catalyze the S-adenosylmethionine (SAM)-dependent methylation of a wide variety of protein and DNA substrates. Methylation of lysine, arginine or cytosine regulates a variety of biological processes including transcriptional activation and gene silencing. Despite extensive studies of the cellular roles of MTs, their quantitative kinetic characterization remains challenging. In the past decade, several assays have been developed to monitor methyl transfer activity utilizing different approaches including radiolabeling, antibodies or mass-spectrometry analysis. However, each approach suffers from different limitation and no easy continuous assay for detection of MT activity exists. We have developed a continuous coupled assay for the general detection of MTs activity. In this assay, the formation of S-adenosylhomocysteine (SAH) product is coupled NAD(P)H oxidation through three enzyme reactions including glutamate dehydrogenase leading to absorbance changes at 340 nm. The utility and versatility of this assay is demonstrated for SET7/9 and SETD6 with peptides and full length protein substrates and for M.HaeIII with a DNA substrate. This study shows a simple and robust assay for the continuous monitoring of MT enzymatic activity. This assay can be used for the determination of steady-state kinetic enzymatic parameters (e.g., k cat and K M) for a wide variety of MTs and can be easily adapted for high-throughput detection of MT activity for various applications.