General acid catalysis by the hepatitis delta virus ribozyme Subha R Das & Joseph A Piccirilli 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. For nearly two decades after the discovery of ribozymes, the view that divalent metal ions mediate RNA catalysis persisted, as all known ribozymes depended on metal ions for their function. The RNA nucleobases, in contrast, were considered poorly suited as catalytic agents. In recent years, structural and functional analyses of small ribozymes have challenged this view: not only do these ribozymes remain active in the absence of divalent metal ions, but their active sites contain nucleobases apparently poised to facilitate reaction chemistry 1–8 . Even the ribosome may rely on direct nucleobase participation to mediate protein synthesis 9,10 . These findings raised questions about the molecular strategies by which nucleobases med- iate catalysis. For the hepatitis delta virus (HDV) ribozyme in particular, it has been proposed that a nucleobase may stabilize charge buildup in the transition state through proton transfer 3–5 . The HDV ribozyme is a self-cleaving RNA motif that occurs in closely related genomic and antigenomic forms during the replication cycle of human HDV 11 . The self-cleavage reaction produces RNA containing 2¢,3¢-cyclic phosphate and 5¢-hydroxyl termini. Crystal- lographic analysis of the self-cleaved motif (product) has shown a critical cytosine (C76 and gC75 as numbered in the antigenomic and genomic forms, respectively) at the active site, leading to the proposal that the nucleobase participates directly in catalysis 1 . Functional analyses have implicated C76 in proton transfer 3–5,11–13 fulfilling one of two kinetically equivalent roles—acting either as a general base 3,4 to deprotonate the 2¢-OH (Fig. 1a) or as a general acid 4,5 to protonate the 5¢-oxygen leaving group (Fig. 1b). Structural data support both possibilities: in the product, the cytosine N3 resides within hydrogen- bonding distance (2.7 A ˚ ) of the 5¢-oxygen atom 1 ; in the inactive C76U mutant ‘precursor,’ equal distances separate N3 (4.6–5.5 A ˚ ) from the 5¢- and 2¢-oxygen atoms 14 . However, global and local conformational changes accompany the cleavage reaction 15–17 , rendering uncertain the relationship between the active-site architectures shown in these structures and the transition-state architecture. Thus neither the structural nor the biochemical data have converged on a coherent mechanistic model for the role of the putative ‘catalytic’ cytosine nucleobase 1,3–5,11,14 . We describe here an approach using a hyperacti- vated substrate that functionally links the ribozyme reaction center to the nucleobase and distinguishes between the kinetically equivalent 18 roles proposed for the active-site nucleobase 3–5,11 . RESULTS Hyperactivated RNA substrate S 5¢-S Chemical perturbation of the leaving group in an enzyme-catalyzed reaction, in combination with enzyme active-site mutations, provides a means of identifying features that contribute to leaving-group stabilization, such as general acid catalysis or hydrogen-bond dona- tion 19–24 . An RNA oligonucleotide containing a 5¢-bridging phosphor- othiolate (5¢-PS) linkage, in which a sulfur atom replaces the 5¢-oxygen atom, offers such a biochemical probe for RNA and protein endonucleases. Under alkaline conditions, a 5¢-PS linkage in RNA reacts nearly 10 5 -fold faster than the native phosphodiester linkages, reflecting the greater acidity of the sulfur leaving group compared with the oxygen leaving group. This hyperactivated 5¢-PS linkage shows susceptibility to base catalysis 25,26 , reacting with a log-linear depen- dence on pH, but lacks susceptibility to general acid catalysis as a consequence of the weak hydrogen bond–accepting ability 27 of the sulfur leaving group. Enzyme mutations or pH changes that disable donation of a hydrogen bond or proton to the leaving group during catalysis (such as those from a general acid) would be expected to affect the cleavage of the 5¢-PS less adversely than cleavage of the Published online 3 May 2005; doi:10.1038/nchembio703 Howard Hughes Medical Institute, Departments of Biochemistry & Molecular Biology and Chemistry, University of Chicago, 5841 S. Maryland Avenue, MC1028, Chicago, Illinois 60637, USA. Correspondence should be addressed to J.A.P. (jpicciri@uchicago.edu). NATURE CHEMICAL BIOLOGY VOLUME 1 NUMBER 1 JUNE 2005 45 ARTICLES © 2005 Nature Publishing Group http://www.nature.com/naturechemicalbiology