Methods of determining SAM-dependent methyltransferase activity using a mutant SAH hydrolase
First Claim
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1. A method for assaying for the activity of a S-adenosylmethionine (SAM)-dependent methyltransferase, comprising:
- a) contacting a SAM-dependent methyltransferase with a substrate of the methyltransferase in the presence of SAM, whereby a methyl group is transferred from the methyltransferase to the substrate and the SAM is converted into S-adenosylhomocysteine (SAH);
b) contacting the resulting SAH with a mutant SAH hydrolase which substantially retains its binding affinity or has enhanced binding affinity for SAH but has attenuated catalytic activity; and
c) detecting binding between the SAH and the mutant SAH hydrolase to detect or determine the presence or amount of the SAH, whereby the activity of the SAM-dependent methyltransferase is assessed.
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Abstract
The present invention relates to compositions and methods for assaying the activity of methyltransferases, such as S-adenosylmethionine (SAM)-dependent methyltransferases. The methods can be used for screening for modulators of such methyltransferases, for identifying substrates and for diagnostics. The methods are amenable for use in high throughput formats. Kits for performing the methods are also provided.
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17 Claims
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1. A method for assaying for the activity of a S-adenosylmethionine (SAM)-dependent methyltransferase, comprising:
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a) contacting a SAM-dependent methyltransferase with a substrate of the methyltransferase in the presence of SAM, whereby a methyl group is transferred from the methyltransferase to the substrate and the SAM is converted into S-adenosylhomocysteine (SAH);
b) contacting the resulting SAH with a mutant SAH hydrolase which substantially retains its binding affinity or has enhanced binding affinity for SAH but has attenuated catalytic activity; and
c) detecting binding between the SAH and the mutant SAH hydrolase to detect or determine the presence or amount of the SAH, whereby the activity of the SAM-dependent methyltransferase is assessed. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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Specification