Lincoln Scott - The pH dependence of hairpin ribozyme catalysis reflects ionization of an active site adenine.

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      Publication Details (including relevant citation   information):

      J Biol   Chem. 2011 May 20;286(20):17658-64. doi:   10.1074/jbc.M111.234906. Epub 2011 Mar 28.

      Abstract:

      Understanding how self-cleaving ribozymes mediate catalysis is   crucial in light of compelling evidence that human and bacterial   gene expression can be regulated through RNA self-cleavage. The   hairpin ribozyme catalyzes reversible phosphodiester bond   cleavage through a mechanism that does not require divalent metal   cations. Previous structural and biochemical evidence implicated   the amidine group of an active site adenosine, A38, in a   pH-dependent step in catalysis. We developed a way to determine   microscopic pK(a) values in active ribozymes based on the   pH-dependent fluorescence of 8-azaadenosine (8azaA). We compared   the microscopic pK(a) for ionization of 8azaA at position 38 with   the apparent pK(a) for the self-cleavage reaction in a fully   functional hairpin ribozyme with a unique 8azaA at position 38.   Microscopic and apparent pK(a) values were virtually the same,   evidence that A38 protonation accounts for the decrease in   catalytic activity with decreasing pH. These results implicate   the neutral unprotonated form of A38 in a transition state that   involves formation of the 5'-oxygen-phosphorus bond.

      Address (URL): http://www.ncbi.nlm.nih.gov/pubmed/21454684