An enzyme-coupled ultrasensitive luminescence assay for protein methyltransferases.
Epigenetic regulation through protein posttranslational modifications is essential in development and disease. Among the key chemical modifications is protein methylation carried out by protein methyltransferases (PMTs). Quantitative and sensitive PMT activity assays can provide valuable tools to investigate PMT functions. Here we developed an enzyme-coupled luminescence assay for S-adenosyl-l-methionine (AdoMet/SAM)-based PMTs. In this assay, S-adenosyl-l-homocystine (AdoHcy/SAH), the by-product of PMT-involved methylation, is sequentially converted to adenine, adenosine monophosphate, and then adenosine 5'-triphosphate (ATP) by 5'-methylthio-adenosine/AdoHcy nucleosidase (MTAN), adenine phosphoribosyl transferase (APRT), and pyruvate orthophosphate dikinase (PPDK), respectively. The resultant ATP can be readily quantified with a luciferin/luciferase kit. This assay is featured for its quantitative linear response to AdoHcy and the ultrasensitivity to 0.3 pmol of AdoHcy. With this assay, the kinetic parameters of SET7/9 methylation were characterized and unambiguously support an ordered mechanism with AdoMet binding as the initial step, followed by the substrate binding and the rate-limiting methylation. The luminescence assay is also expected to be generally applicable to many other AdoMet-dependent enzymes. In addition, the mix-and-measure 96-/384-well format of our assay makes it suitable for automation and high throughput. Our enzyme-coupled luminescence assay, therefore, represents a convenient and ultrasensitive approach to examine methyltransferase activities and identify methyltransferase inhibitors.
Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.
Glorymar
G
Ibáñez
Jamie L
JL
McBean
Yaritzy M
YM
Astudillo
Minkui
M
Luo
D057075:Enzyme Assays / Q000379:methods*; D006649:Histocompatibility Antigens / Q000378:metabolism; D011495:Histone-Lysine N-Methyltransferase / Q000378:metabolism; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D011496:Protein Methyltransferases / Q000378:metabolism*; D012435:S-Adenosylhomocysteine / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism*; D012680:Sensitivity and Specificity
D006649:Histocompatibility Antigens; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D011496:Protein Methyltransferases; C445405:EHMT2 protein, human; D011495:Histone-Lysine N-Methyltransferase; C540312:SETD7 protein, human