RNA Nucleobase Is Key to Catalysis

ENZYMOLOGY

Chemists have finally produced functional evidence that RNA nucleobases--once thought to be poorly suited as catalytic agents--can directly mediate proton transfer in RNA enzymes.

RNA enzymes (ribozymes) were thought to rely solely on bound divalent metal ions for catalytic activity. More recent studies, however, have shown that certain ribozymes operate even in the absence of divalent metal ions. In addition, X-ray crystallography has revealed that certain ribozymes contain active-site nucleobases that appear to be positioned to participate in catalysis.

These observations have led scientists to suspect that, in some ribozymes, active-site nucleobases might directly participate in catalysis as general acids or general bases, just as protein side chains such as histidine do, notes Martha J. Fedor of Scripps Research Institute. But there's been no simple way to test this hypothesis.

Now, Subha R. Das and Joseph A. Piccirilli of the University of Chicago and Howard Hughes Medical Institute have developed such a test and have used it to provide the first evidence that an RNA active-site nucleobase plays a direct catalytic role in the hepatitis delta virus (HDV) ribozyme, a model ribozyme that cleaves its RNA substrate at a specific phosphodiester bond (Nat. Chem. Biol., published online May 3, dx.doi.org/10.1038/nchembio703). Structural studies of the HDV ribozyme previously revealed that a cytosine in its active site seems poised to assist catalysis, but this residue's role remained uncertain, Piccirilli notes.

To test whether this nucleobase acts as a general acid (a proton donor) during the HDV catalytic cycle, he and Das synthesized a modified RNA substrate in which the phosphodiester (P-O) linkage that the ribozyme normally cuts is replaced with a phosphorothiolate (P-S) linkage. The cleavage of this P-S substrate is less dependent on protonation from a general acid than is the cleavage of the natural P-O substrate. Piccirilli and Das find that an HDV enzyme containing a modified cytosine that can't donate a proton can't cleave the natural P-O substrate but readily cleaves the modified P-S one.

They also show that the ribozyme cleaves its natural substrate more slowly with increasing pH, when there is less protonated cytosine available. Taken together, these results suggest that cytosine indeed acts as a general acid, Piccirilli says. "RNA nucleobases may function in a manner analogous to that of catalytic histidine residues in protein enzymes," he adds.

Piccirilli and Das's strategy can be used to test for general acids in the active sites of other ribozymes, too, Fedor notes. "Their strategy illustrates a general way to link specific active-site functional groups to particular bond-making or bond-breaking steps in a phosphodiester cleavage reaction," she says.