D1R, an active subdomain of the large subunit of vaccinia virus mRNA capping enzyme possessing ATPase, RNA 5`-triphosphatase, and guanylyltransferase activities, was expressed in Escherichia coli and shown to be functionally equivalent to the heterodimeric enzyme (Myette, J. R., and Niles, E. G. (1996) J. Biol. Chem. 271, 11936-11944). A detailed characterization of the phosphohydrolytic activities of D1R demonstrates that, in addition to ATPase and RNA 5`-triphosphatase activities, the capping enzyme also possesses a general nucleoside triphosphate phosphohydrolase activity that lacks a preference for the nucleoside base or sugar. Nucleoside triphosphate and mRNA saturation kinetics are markedly different, with RNA exhibiting a K and turnover number 100- and 10-fold less, respectively, than those values measured for any NTP. The linear competitive inhibition of RNA 5`-triphosphatase activity by ATP, and the relative manner by which both ATPase and RNA 5`-triphosphatase activities are inhibited by specific oligonucleotides, kinetically demonstrate that each activity is carried out at a common active site. Direct UV photo-cross-linking of either P-radiolabeled ATP or 23-mer triphosphorylated RNA, followed by cyanogen bromide cleavage of the photo-linked enzyme, localizes the major binding site for both ATP and RNA to a region between amino acids 1 and 221. The inability of ATP to competitively inhibit either EGMP formation or the transfer of GMP to RNA kinetically differentiates the phosphohydrolase active site from the guanylyltransferase active site.

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