Potent Inhibition of Ribonuclease A by Oligo(vinylsulfonic Acid)

Bryan D Smith,Matthew B Soellner,Ronald T Raines

doi:10.1074/jbc.m301852200

Bryan D Smith, Matthew B Soellner + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m301852200

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Abstract

Ribonuclease A (RNase A) can make multiple contacts with an RNA substrate. In particular, the enzymatic active site and adjacent subsites bind sequential phosphoryl groups in the RNA backbone through Coulombic interactions. Here, oligomers of vinylsulfonic acid (OVS) are shown to be potent inhibitors of RNase A that exploit these interactions. Inhibition is competitive with substrate and has Ki = 11 pm in assays at low salt concentration. The effect of salt concentration on inhibition indicates that nearly eight favorable Coulombic interactions occur in the RNase A.OVS complex. The phosphonic acid and sulfuric acid analogs of OVS are also potent inhibitors although slightly less effective. OVS is also shown to be a contaminant of MES and other buffers that contain sulfonylethyl groups. Oligomers greater than nine units in length can be isolated from commercial MES buffer. Inhibition by contaminating OVS is responsible for the apparent decrease in catalytic activity that has been observed in assays of RNase A at low salt concentration. Thus, OVS is both a useful inhibitor of RNase A and a potential bane to chemists and biochemists who use ethanesulfonic acid buffers.

Highlights

RNA is the least stable of the biopolymers that effect information transfer in biology [1]
While studying Ribonuclease A (RNase A) catalysis as a function of salt concentration, we found that a contaminant in common biological buffers was a potent inhibitor in solutions of low salt concentrations [10]
An aliquot of inhibitor (I) dissolved in the Purification of Inhibitor from a Commercial Buffer—Previously, we reported that a contaminant in MES buffer inhibits catalysis by RNase A, especially in solutions of low salt concentration [10]

Summary

Introduction

RNA is the least stable of the biopolymers that effect information transfer in biology [1]. Similar to most known ribonucleases, RNase A can make multiple contacts with an RNA substrate (Fig. 1). The enzymatic active site and adjacent subsites bind sequential phosphoryl groups in the RNA backbone through Coulombic inter-. A pyrophosphatelinked oligonucleotide (pdUppA-3Ј-p), which occupies three subsites, is the tightest known small-molecule inhibitor of RNase A [4]. Nature uses this strategy to inhibit RNase A and its homologs. The 50-kDa ribonuclease inhibitor protein forms a tight 1:1 complex with RNase A (Kd ϳ10Ϫ14 M) [6], chelating all of its phosphoryl group binding subsites [7]. Pyrophosphate-linked oligonucleotides are susceptible to hydrolysis [8], and ribonuclease inhibitor is readily inactivated by oxidation [9]

Results

Discussion

Conclusion