Ribonucleic general acidScott A Strobel

Ribozymes are enzymes comprised of RNA that fold into three-dimensional structures and that catalyze chemical reactions central to all cells. A new study shows that ribozymes, like proteins, may use general acid catalysis as part of their catalytic toolkit.

As a biological catalyst, RNA would appear to be underpowered, seemingly outgunned by the diverse chemical side chains available to proteins. But in this first issue of Nature Chemical Biology, Das and Piccirilli1 dem-onstrate that RNA has more catalytic tricks at its disposal than was originally thought when catalytic RNA was discovered. Using a clever chemogenetic suppression analysis, the authors provide compelling evidence that an RNA enzyme uses general acid catalysis. Their approach, which merges chemical substitution with enzyme kinetics, provides an elegant and potentially general method to test whether a particular functional group acts as a general acid or general base in enzyme catalysis.

General acid or general base catalysis is used by enzymes to facilitate the proton transfers required in biochemical transfor-mations. This catalytic mechanism requires that enzymes position active-site acidic or basic side chains with appropriate pKa val-ues near the substrate. This possibility was considered unlikely ten years ago because the pKas of the nucleobases were viewed as being outside the catalytically useful range2. Recent evidence has suggested that RNA can perturb the pKas of its nucleobase functional groups sufficiently close to neutrality that RNA could use general acid or general base catalysis3,4. The possibility of general acid-base catalysis by RNA has been most exten-sively explored in the hepatitis delta virus (HDV) ribozyme, a cis-acting enzyme that cleaves its RNA genome into unit-sized lin-ear fragments as part of rolling-circle replica-tion5. At the cleavage site, a 2′-OH attacks the

S linkage shows strong lability toward basic conditions in the uncatalyzed reaction, but is relatively unaffected by acidic pH. Thus, this substitution provides a straightforward means to distinguish between the roles attributed to C76. Das and Piccirilli reported that a C76U mutant ribozyme, which cannot cleave the normal P-O bond, was able to cleave the P-S substrate at wild-type levels. Furthermore, the addition of imidazole, which partially rescues the C76U mutation for P-O bond cleavage, had no effect on the rate of P-S bond cleavage. These results strongly implicate C76 action upon the O5′ leaving group.

The authors further refined their con-clusions by examining the effect of chemi-cal substitutions of C76 on cleavage of the hyperactivated substrate. First, they elimi-nated the proton transfer capability of C76 by replacing N3 with a carbon (Fig. 1). Consistent with a general acid mechanism involving the N3 imino group, they observed complete loss of activity for P-O bond cleav-

scissile phosphate to liberate a 5′-OH, with the 2′-OH losing a proton and the O5′ leav-ing group accepting one during the course of the reaction (Fig. 1). A cytidine (C76 in the antigenomic form of the enzyme studied by Das and Piccirilli) is essential to the self-cleavage reaction. Crystal structures of the cleaved and uncleaved forms of the genomic HDV ribozymes have shown that the equiva-lent cytidine is reasonably positioned to act catalytically6,7. What remained ambiguous was whether the cytidine acts as a general acid or as a general base.

Das and Piccirilli used a series of chemi-cal substitutions at residues surrounding the HDV ribozyme active center. Most impor-tantly, they replaced the O5' leaving group with a hyperactivated 5′-bridging sulfur group (5′-PS, Fig. 1), a substitution that eliminates the need for a general acid catalysis during bond cleavage. The 5′-sulfur is an excellent leaving group but is not a particularly good hydrogen-bond acceptor. As a result the P-

Scott A. Strobel is in the Department of Molecular Biophysics & Biochemistry, Yale University, 260 Whitney Avenue, New Haven, Connecticut 06520-8114, USA.e-mail: scott.strobel@yale.edu.

Figure 1 General acid catalysis by the HDV ribozyme. (a) Proposed mechanism for general acid catalysis of phosphodiester cleavage by the general acid C76 in the HDV ribozyme. (b) Das and Piccirilli probed the role of C76 through functional-group substitutions in the phosphodiester backbone (X) and the cytosine base (Y and Z).

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age, but virtually no effect for P-S bond cleavage. Second, they enhanced the acid-ity of the cytidine N3 by replacing C6 with a nitrogen atom (Fig. 1) and explored the pH dependence of P-S and P-O bond cleav-age. Divergence in the pH-rate profile of the functional-group mutant provided evidence that deprotonation of C76 N3 is inhibitory. The data fully support the mechanistic pro-posal originally advanced by Bevilacqua and coworkers, who suggested that the active-site cytidine acts as a general acid4.

These compelling biochemical data seem to contradict conclusions derived from crystal structures of the uncleaved genomic HDV ribozyme. In a previous report, Ke et al. trapped the genomic form of the ribozyme in an unreactive state7. They observed that the C75 N3 (corresponding to C76 in the ribozyme studied by Das and Piccirilli) was closest to the O2′ nucleophile, suggestive of general base catalysis by C75. It is conceiv-able that the mechanisms of the genomic and antigenomic ribozymes differ, but this seems unlikely. Das and Piccirilli do not attempt

to reconcile their biochemical data with the previous structural results, arguing simply that the different conformations observed for the substrate and product states render the structures ambiguous. The conclusions of Das and Piccirilli necessitate that the chemical details of the transition state must differ from those inferred from the structural studies of Ke et al. Specifically, the transi-tion state must have a conformation closer to the product than the substrate structures. It may be necessary to obtain structures of transition-state mimics before a convergence between the biochemical and structural results can be achieved.

The HDV ribozyme precedent argues that other catalytic RNAs could use general acid catalysis. For example, a conserved adenosine in the VS ribozyme and a conserved guano-sine in the hairpin ribozyme are both cru-cial for the RNA cleavage reaction and each has been proposed to act as a general acid or general base8,9. Peptide bond formation by the ribosome may also proceed by general acid-base catalysis, although currently the best

candidate for catalytic participation is not the nucleobase of the ribosome but rather the 2′-OH at the end of the tRNA substrate10. This report by Das and Piccirilli provides the means needed to characterize such systems; the ability to ask fine-tuned mechanistic questions using chemically manipulated substrates provides a powerful synergy at the interface of chemistry and biology.

1. Das, S.R. & Piccirilli, J.A. Nat. Chem. Biol 1, 45–52 (2005).

2. Saenger, W. Principles of Nucleic Acid Structure (Springer, New York, 1984).

3. Perrotta, A.T., Shih, I. & Been, M.D. Science 286, 123–126 (1999).

4. Nakano, S., Chadalavada, D.M. & Bevilacqua, P.C. Science 287, 1493–1497 (2000).

5. Shih, I. & Been, M.D. Annu. Rev. Biochem. 71, 887–917 (2002).

6. Ferre-D’Amare, A.R., Zhou, K. & Doudna, J.A. Nature 395, 567–574 (1998).

7. Ke, A., Zhou, K., Ding, F., Cate, J.H. & Doudna, J.A. Nature 429, 201–205 (2004).

8. Lafontaine, D., Wilson, T., Zhao, Z. & Lilley, D.M. J. Mol. Biol. 323, 23–24 (2002).

9. Rupert, P.B. & Ferre-D’Amare, A.R. Nature 410, 780–786 (2001).

10. Weinger, J.S., Parnell, K.M., Dorner, S., Green, R. & Strobel, S.A. Nat. Struct. Mol. Biol. 11, 1101–1106 (2004).

Pulling NO out of thin airThomas G Spiro

Nitric oxide signaling requires that the heme of soluble guanylate cyclase bind NO preferentially to O2. Engineering of the enzyme’s active site reveals a molecular basis for NO binding selectivity.

Thomas G. Spiro is in the Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA.e-mail: spiro@princeton.edu

Nitric oxide (NO) has a critical role in eukary-otic physiology. Signaling is initiated by NO binding to the heme protein soluble guanyl-ate cyclase (sGC), which activates production of cyclic guanosine monophosphate (cGMP). But heme groups can bind O2 as well as NO, and ambient O2 levels are typically 1,000-fold higher than those of NO. How does sGC avoid being swamped by O2? On p. 53 of this issue, Boon et al.1 show that keeping hydrogen-bond donors out of the active site is an important part of the answer.

The heme group is nature’s main receptor for the gases CO, NO and O2, all of which play vital biological roles. These diatomic molecules bind to the heme iron atom by accepting iron electrons into their empty (π*) orbitals, an interaction known as back-bonding. But how

does the heme protein choose among these gases, and how does it subsequently control their chemistry? The answer must lie in addi-tional interactions between the heme adduct and the surrounding protein. One example in which it is known how heme proteins control ligand specificity is the case of the O2-binding proteins, myoglobin and hemoglobin, which must keep themselves from being poisoned by the body’s own CO (ref. 2).

The affinity of heme for CO is 20,000-fold greater than that for O2, but this preference is lowered by a factor of 1,000 in the globins. Because the preferred geometry is linear for the CO adduct but bent for the O2 adduct, steric hindrance from a distal histidine resi-due, which is located directly over the heme iron atom in the globins, had been thought to discriminate against CO. At the same time, hydrogen bonding from the same distal histi-dine could discriminate in favor of O2, because the transfer of negative charge from the heme to the bound ligand is greater for O2 than for CO; this hydrogen bond is apparent in crystal

structures. The contribution of the histidine to ligand specificity was resolved by replac-ing the distal histidine with a nonpolar but approximately isosteric residue (valine, leucine or isoleucine), which essentially abolished the 1,000-fold discrimination3, mainly by decreas-ing the O2 affinity. The hydrogen-bond effect is estimated to account for at least 85% of the discrimination2, a conclusion supported by ab initio electronic structure computations4.

The biological scope of ligand-binding heme proteins has recently been expanded far beyond O2 transport and activation by the discovery of sensor proteins that turn on gene activation or an enzymatic activity in response to binding of O2, NO or CO (ref. 5). sGC is the most prominent of these, being the uni-versal receptor for eukaryotic NO signaling6. However, the factors that allow sGC to distin-guish between NO and O2 were unknown.

A clue to the origin of NO specificity came from the discovery that gene sequences homo-logous to those for the sGC heme-binding domain are also widespread among prokary-

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