A coupled fluorescent assay for histone methyltransferases.
Histone methyltransferases (HMTs) catalyze the S-adenosylmethionine (AdoMet)-dependent methylation of lysines and arginines in the nucleosomal core histones H3 and H4 and the linker histone H1b. Methylation of these residues regulates either transcriptional activation or silencing, depending on the residue modified and its degree of methylation. Despite an intense interest in elucidating the functions of HMTs in transcriptional regulation, these enzymes have remained challenging to quantitatively assay. To characterize the substrate specificity of HMTs, we have developed a coupled-fluorescence-based assay for AdoMet-dependent methyltransferases. This assay utilizes S-adenosylhomocysteine hydrolase (SAHH) to hydrolyze the methyltransfer product S-adenosylhomocysteine (AdoHcy) to homocysteine (Hcy) and adenosine (Ado). The Hcy concentration is then determined through conjugation of its free sulfhydryl moiety to a thiol-sensitive fluorophore. Using this assay, we have determined the kinetic parameters for the methylation of a synthetic histone H3 peptide (corresponding to residues 1-15 of the native protein) by Schizosaccharomyces pombe CLR4, an H3 Lys-9-specific methyltransferase. The fluorescent SAHH-coupled assay allows rapid and facile determination of HMT kinetics and can be adapted to measure the enzymatic activity of a wide variety of AdoMet-dependent methyltransferases.
Collazo
Evys
E
Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109-0606, USA.
Couture
Jean-François
JF
Bulfer
Stacie
S
Trievel
Raymond C
RC
D043383:Adenosylhomocysteinase / Q000378:metabolism; D003374:Coumarins / Q000737:chemistry; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000032:analysis*; D006710:Homocysteine / Q000032:analysis; D007700:Kinetics; D008745:Methylation; D011496:Protein Methyltransferases; D011484:Protein-Arginine N-Methyltransferases / Q000032:analysis; D012568:Schizosaccharomyces / Q000378:metabolism; D048229:Sulfolobus solfataricus / Q000201:enzymology
D003374:Coumarins; D005456:Fluorescent Dyes; D006710:Homocysteine; C030860:N-(4-(7-diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide; D000076983:Histone Methyltransferases; D011496:Protein Methyltransferases; D011484:Protein-Arginine N-Methyltransferases; D011495:Histone-Lysine N-Methyltransferase; D043383:Adenosylhomocysteinase