General acid catalysis by the hepatitis delta virus ribozyme

Recent crystallographic and functional analyses of RNA enzymes have raised the possibility that the purine and pyrimidine nucleobases may function as general acid-base catalysts. However, this mode of nucleobase-mediated catalysis has been difficult to establish unambiguously. Here, we used a hyperactivated RNA substrate bearing a 5'-phosphorothiolate to investigate the role of a critical cytosine residue in the hepatitis delta virus ribozyme. The hyperactivated substrate specifically suppressed the deleterious effects of cytosine mutations and pH changes, thereby linking the protonation of the nucleobase to leaving-group stabilization. We conclude that the active-site cytosine provides general acid catalysis, mediating proton transfer to the leaving group through a protonated N3-imino nitrogen. These results establish a specific role for a nucleobase in a ribozyme reaction and support the proposal that RNA nucleobases may function in a manner analogous to that of catalytic histidine residues in protein enzymes.