Chlorella virus RNA triphosphatase. Mutational analysis and mechanism of inhibition by tripolyphosphate

Abstract

Chlorella virus RNA triphosphatase (cvRtp1) is the smallest member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, protozoa, poxviruses, and baculoviruses. The primary structure of cvRtp1 is more similar to that of the yeast RNA triphosphatase Cet1 than it is to the RNA triphosphatases of other DNA viruses. To evaluate the higher order structural similarities between cvRtp1 and the fungal enzymes, we performed an alanine scan of individual residues of cvRtp1 that were predicted, on the basis of the crystal structure of Cet1, to be located at or near the active site. Twelve residues (Glu(24), Glu(26), Asp(64), Arg(76), Lys(90), Glu(112), Arg(127), Lys(129), Arg(131), Asp(142), Glu(163), and Glu(165)) were deemed essential for catalysis by cvRtp1, insofar as their replacement by alanine reduced phosphohydrolase activity to <5% of the wild-type value. Structure-activity relationships were elucidated by introducing conservative substitutions at the essential positions. The mutational results suggest that the active site of cvRtp1 is likely to adopt a tunnel fold like that of Cet1 and that a similar constellation of side chains within the tunnel is responsible for metal binding and reaction chemistry. Nonetheless, there are several discordant mutational effects in cvRtp1 versus Cet1, which suggest that different members of the phosphohydrolase family vary in their reliance on certain residues within the active site tunnel. We found that tripolyphosphate and pyrophosphate were potent competitive inhibitors of cvRtp1 (K(i) = 0.6 microm tripolyphosphate and 2.4 microm pyrophosphate, respectively), whereas phosphate had little effect. cvRtp1 displayed a weak intrinsic tripolyphosphatase activity (3% of its ATPase activity) but was unable to hydrolyze pyrophosphate.