The pH Dependence of Hairpin Ribozyme Catalysis Reflects Ionization of an Active Site Adenine

Joseph W Cottrell,Lincoln G Scott,Martha J Fedor

doi:10.1074/jbc.m111.234906

Joseph W Cottrell, Lincoln G Scott + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m111.234906

Copy DOI

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.

Highlights

Synthesis of 8-azaadenosine 5Ј-monophosphate (8azaAMP)— 8azaAMP was prepared using an in vitro enzymatic synthesis method beginning with the C5 phosphorylation of ribose by ribokinase to give ribose 5-phosphate, which was further phosphorylated at C1 by 5-phosphoD-ribosyl-␣-1-pyrophosphate synthetase, forming 5-phospho-D-ribosyl-␣-1-pyrophosphate (Fig. 3)
Due to the lack of a catalytically active 8azaATP pyrophosphatase, the second step of the synthesis involved essentially the reverse reaction to discharge the 8azaATP to 8azaAMP
High resolution structures of Hairpin ribozymes (Hp Rz) complexes with transition state mimics place G8 and A38 near the reactive phosphate, positioned like the two histidines in RNase A that mediate general acid-base catalysis of the same reaction (Fig. 1A) (6 –9), making the protonation state of these nucleobases the focus of considerable interest

Summary

Functional and Microscopic pKa Correspondence in a Ribozyme

Increase in activity with increasing pH could be observed if donation of a proton to the 5Ј-oxygen by the protonated form of adenosine and withdrawal of a proton from the 2Ј-oxygen by hydroxide ion occurred in the transition state or if the reaction involved unprotonated adenosine alone [10, 20]. Molecular dynamics simulations support a model in which active site interactions provide electrostatic stabilization of the electronegative transition state without direct participation of RNA functional groups in proton transfer [23]. We set out to gain a better understanding of how RNA functional groups can participate in catalysis and to distinguish among alternative roles for A38 in the Hp Rz mechanism by determining the protonation state of A38(N1) directly in a functional ribozyme. These results contradict the idea that the cationic protonated form of A38 is essential for catalysis Instead, they point to a view of the Hp Rz transition state in which the neutral unprotonated form of A38 facilitates formation of the 5Ј-oxygen–phosphorus bond

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION