Direct photolinkage of GTP to the vaccinia virus mRNA (guanine-7-) methyltransferase GTP methyl acceptor site.
Direct UV photolinkage of [alpha-32P]GTP to the methyl acceptor site of the vaccinia virus (guanine-7-) methyltransferase was attempted in order to identify the GTP binding region of this enzyme. Low-efficiency photolinkage of GTP to the carboxyl terminal domain of the large subunit, D1R498-844, was achieved and shown to be specific by several criteria. The half-saturation value for GTP was determined to be 35 microM which is equivalent to the catalytic Km for the substrate. GTP photolinkage was shown to be inhibited by GpppA, a substrate for the methyltransferase reaction, better than GMepppA, the reaction product. The addition of MgCl2, known to prevent GTP from serving as a methyl group acceptor in this reaction, was found to eliminate GTP photolinkage. Finally, AdoHcy, a potent product inhibitor of AdoMet binding, failed to inhibit GTP photolinkage, demonstrating that GTP was not linked to the AdoMet binding site. Chemical cleavage of the GTP-labeled enzyme permitted the identification of multiple radioactive peptides, demonstrating the existence of multiple interaction sites in the carboxyl terminal domain of the D1R subunit. The addition of the small D12L subunit has been shown to activate the (guanine-7-) methyltransferase activity in D1R498-844 30-50-fold. The efficiency of GTP photolinkage to the isolated D1R498-844 domain, however, was found to be only marginally effected by the addition of the D12L subunit, demonstrating that this enhancement of mRNA (guanine-7-) methyltransferase activity mediated by D12L was not achieved by altering the strength of GTP binding.
Niles
E G
EG
Biochemistry Department, School of Medicine, State University of New York, Buffalo 14214.
Christen
L
L
Higman
M A
MA
D001665:Binding Sites; D001667:Binding, Competitive; D004926:Escherichia coli / Q000201:enzymology; D006147:Guanine / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D015636:Magnesium Chloride / Q000494:pharmacology; D008780:Methyltransferases / Q000378:metabolism*; D010777:Photochemistry; D012333:RNA, Messenger / Q000378:metabolism*; D012435:S-Adenosylhomocysteine / Q000494:pharmacology; D012436:S-Adenosylmethionine / Q000378:metabolism; D014466:Ultraviolet Rays; D014616:Vaccinia virus / Q000235:genetics*
D012333:RNA, Messenger; D015636:Magnesium Chloride; D006147:Guanine; D012436:S-Adenosylmethionine; D006160:Guanosine Triphosphate; D012435:S-Adenosylhomocysteine; D008780:Methyltransferases