A general liquid chromatography/mass spectroscopy-based assay for detection and quantitation of methyltransferase activity.
Methyltransferases form a large class of enzymes, most of which use S-adenosylmethionine as the methyl donor. In fact, S-adenosylmethionine is second only to ATP in the variety of reactions for which it serves as a cofactor. Several methods to measure methyltransferase activity have been described, most of which are applicable only to specific enzymes and/or substrates. In this work we describe a sensitive liquid chromatography/mass spectroscopy-based methyltransferase assay. The assay monitors the conversion of S-adenosylmethionine to S-adenosylhomocysteine and can be applied to any methyltransferase and substrate of interest. We used the well-characterized enzyme catechol O-methyltransferase to demonstrate that the assay can monitor activity with a variety of substrates, can identify new substrates, and can be used even with crude preparation of enzyme. Furthermore, we demonstrate the utility of the assay for kinetic characterization of enzymatic activity.
Salyan
Mary Ellen K
ME
Department of Discovery Analytical Sciences, Bristol-Myers Squibb Co., 311 Pennington-Rocky Hill Rd., Pennington, NJ 08534, USA.
Pedicord
Donna L
DL
Bergeron
Laurie
L
Mintier
Gabe A
GA
Hunihan
Lisa
L
Kuit
Kathy
K
Balanda
Lynn A
LA
Robertson
Barbara J
BJ
Feder
John N
JN
Westphal
Ryan
R
Shipkova
Petia A
PA
Blat
Yuval
Y
D000595:Amino Acid Sequence; D002394:Catechol O-Methyltransferase / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D002853:Chromatography, Liquid; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D012435:S-Adenosylhomocysteine / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D012435:S-Adenosylhomocysteine; D002394:Catechol O-Methyltransferase