An enzyme-coupled continuous spectrophotometric assay for S-adenosylmethionine-dependent methyltransferases.
Modification of small molecules and proteins by methyltransferases affects a wide range of biological processes. Here, we report an enzyme-coupled continuous spectrophotometric assay to quantitatively characterize S-adenosyl-L-methionine (AdoMet/SAM)-dependent methyltransferase activity. In this assay, S-adenosyl-L-homocysteine (AdoHcy/SAH), the transmethylation product of AdoMet-dependent methyltransferases, is hydrolyzed to S-ribosylhomocysteine and adenine by recombinant S-adenosylhomocysteine/5'-methylthioadenosine nucleosidase (SAHN/MTAN, EC 3.2.2.9). Subsequently, adenine generated from AdoHcy is further hydrolyzed to hypoxanthine and ammonia by recombinant adenine deaminase (EC 3.5.4.2). This deamination is associated with a decrease in absorbance at 265 nm that can be monitored continuously. Coupling enzymes are recombinant and easily purified. The utility of this assay was shown using recombinant rat protein arginine N-methyltransferase 1 (PRMT1, EC 2.1.1.125), which catalyzes the mono- and dimethylation of guanidino nitrogens of arginine residues in select proteins. Using this assay, the kinetic parameters of PRMT1 with three synthetic peptides were determined. An advantage of this assay is the destruction of AdoHcy by AdoHcy nucleosidase, which alleviates AdoHcy product feedback inhibition of S-adenosylmethionine-dependent methyltransferases. Finally, this method may be used to assay other enzymes that produce AdoHcy, 5'-methylthioadenosine, or compounds that can be cleaved by AdoHcy nucleosidase.
Dorgan
Kathleen M
KM
Department of Chemistry, Washington State University, Pullman, WA 99164, USA.
Wooderchak
Whitney L
WL
Wynn
Donraphael P
DP
Karschner
Erin L
EL
Alfaro
Joshua F
JF
Cui
Yinqiu
Y
Zhou
Zhaohui Sunny
ZS
Hevel
Joan M
JM
D000619:Aminohydrolases / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D019271:Hypoxanthine / Q000032:analysis; D007700:Kinetics; D008780:Methyltransferases / Q000032:analysis*; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D011484:Protein-Arginine N-Methyltransferases / Q000032:analysis; D051381:Rats; D012435:S-Adenosylhomocysteine / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism*; D013053:Spectrophotometry / Q000379:methods*
D019271:Hypoxanthine; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D008780:Methyltransferases; D011484:Protein-Arginine N-Methyltransferases; D009699:N-Glycosyl Hydrolases; C019606:adenosylhomocysteine nucleosidase; D000619:Aminohydrolases; C018774:adenine deaminase