Inhibition of human poly(A)-specific ribonuclease (PARN) by purine nucleotides: kinetic analysis

Abstract

Poly(A)-specific ribonuclease (PARN) is a cap-interacting and poly(A)-specific 30 -exoribonuclease that efficiently degrades mRNA poly(A) tails. Based on the enzyme's preference for its natural substrates, we examined the role of purine nucleotides as potent effectors of human PARN activity. We found that all purine nucleotides tested can reduce poly(A) degradation by PARN. Detailed kinetic analysis revealed that RTP nucleotides behave as non-competitive inhibitors while RDP and RMP exhibit competitive inhibition. Mg 2 þ which is a catalytically important mediator of PARN activity can release inhibition of RTP and RDP but not RMP. Although many strategies have been proposed for the regulation of PARN activity, very little is known about the modulation of PARN activity by small molecule effectors, such as nucleotides. Our data imply that PARN activity can be modulated by purine nucleotides in vitro, providing an additional simple regulatory mechanism.

Dissimilar Roles of the Four Conserved Acidic Residues in the Thermal Stability of Poly(A)-Specific Ribonuclease

“…Considering that PARN is an allosteric enzyme [ 29 ], it is important to find that the Mg 2+ coordination could induce conformational changes in the other domains of PARN, which might contribute to the allosteric regulation of PARN. This conclusion is quite consistent with the previous observations by Balatsos et al ., which showed that the coordination of Mg 2+ could release the non-competitive nucleotide inhibitors of PARN [ 30 ]. Our findings is also consistent with that previously observed by Dupureur and Dominguez, which showed that the mutations in the active site of Pvu II endonuclease resulted in significant changes in the conformation and stability of the enzyme [ 13 ].…”

AtHESPERIN: a novel regulator of circadian rhythms with poly(A)-degrading activity in plants

“…The enzymatic activity of recombinant AtHESP was determined by a colorimetric assay as described with some modifications. [24][25][26] Briefly, the reactions were performed with 0.1-0.15 nM recombinant AtHESP in 1 ml reactions. The absorbance of poly(A), poly(U), poly(C) or poly(G) (all from Sigma-Aldrich) was measured at appropriate wavelengths [poly(A), 662 nm; poly(U), 663 nm; poly(G), 665 nm; poly(C), 667 nm].…”

Competitive Inhibition of Human Poly(A)-Specific Ribonuclease (PARN) by Synthetic Fluoro-Pyranosyl Nucleosides

“…Methylene blue buffer was prepared by dissolving 1. (19). The final reaction volume was 100 μL, and the reaction was performed at 30°C for 5-10 min.…”

“…Furthermore, it has been shown that natural purine nucleotides also inhibit PARN activity (18,19). More specifically, we have shown previously that RTP nucleotides inhibit the enzyme noncompetitively, while RDP and RMP act in a competitive manner (19).…”

“…Aminoglycosides are antibiotics widely used in clinical practice and this inhibition could explain the toxicity side effects that these antibiotics exhibit during treatment (17). Furthermore, it has been shown that natural purine nucleotides also inhibit PARN activity (18,19). More specifically, we have shown previously that RTP nucleotides inhibit the enzyme noncompetitively, while RDP and RMP act in a competitive manner (19).…”