Direct photolabeling of the EcoRII methyltransferase with S-adenosyl-L-methionine.
Ultraviolet irradiation of EcoRII methyltransferase in the presence of its substrate, S-adenosyl-L-methionine (AdoMet), results in the formation of a stable enzyme-substrate adduct. This adduct can be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after irradiation of the enzyme in the presence of either [methyl-3H]AdoMet or [35S]AdoMet. The extent of photolabeling is low. Under optimal conditions, 4.5 pmol of [3H]AdoMet is incorporated into 100 pmol of enzyme. Use of the 8-azido derivative of AdoMet as the photolabeling substrate increases the incorporation by approximately 2-fold. However, this adduct, unlike the one formed with AdoMet, is not stable when treated with thiol reagents or precipitated with trichloroacetic acid. A catalytically active conformation of the enzyme is needed for AdoMet photolabeling. Heat-inactivated enzyme or proteins for which AdoMet is not a substrate or cofactor do not undergo adduct formation. Two other methyltransferases, MspI and dam methylases are also shown to form adducts with AdoMet upon UV irradiation. The binding constant of the EcoRII methyltransferase for AdoMet determined with the photolabeling reaction is 11 microM, which is similar to the binding constant of 9 microM previously reported (Friedman, S. (1986) Nucleic Acids Res. 14, 4543-4556). The AdoMet analogs S-adenosyl-L-homocysteine (Ki = 0.83 microM) and sinefungin (Ki = 4.3 microM) are effective inhibitors of photolabeling, whereas S-adenosyl-D-homocysteine (Ki = 46 microM) is a poor inhibitor. These experiments indicate that AdoMet becomes covalently bound at the AdoMet-binding site on the enzyme molecule. The EcoRII methyltransferase-AdoMet adduct is very stable and could be used to identify the AdoMet-binding site on DNA methyltransferases.
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Department of Pharmacology, State University of New York Health Science Center, Brooklyn 11203.
Friedman
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D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000345:Affinity Labels; D001386:Azides; D001483:Base Sequence; D011232:Chemical Precipitation; D004269:DNA, Bacterial / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000378:metabolism / Q000528:radiation effects; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli; D007700:Kinetics; D008623:Mercaptoethanol; D008745:Methylation; D010777:Photochemistry; D011487:Protein Conformation; D012435:S-Adenosylhomocysteine / Q000494:pharmacology; D012436:S-Adenosylmethionine / Q000031:analogs & derivatives / Q000378:metabolism; D013379:Substrate Specificity; D014238:Trichloroacetic Acid; D014466:Ultraviolet Rays
D000345:Affinity Labels; D001386:Azides; D004269:DNA, Bacterial; D014238:Trichloroacetic Acid; D008623:Mercaptoethanol; D012436:S-Adenosylmethionine; C037207:8-azido-S-adenosylmethionine; D012435:S-Adenosylhomocysteine; C029528:DNA modification methylase EcoRII; D015257:DNA-Cytosine Methylases; D000241:Adenosine; D004229:Dithiothreitol; C006235:sinefungin