Magnesium-binding studies reveal fundamental differences between closely related RNA triphosphatases.
The Chlorella virus RNA triphosphatase (cvRTPase) is involved in the formation of the RNA cap structure found at the 5'-end of the viral mRNAs and requires magnesium ions to mediate its catalytic activity. To extend our studies on the role of metal ions in phosphohydrolysis, we have used a combination of fluorescence spectroscopy, circular dichroism, denaturation studies and thermodynamic analyses to monitor the binding of magnesium ions to the cvRTPase. Using these techniques, the thermodynamic forces responsible for the interaction of metal ions with an RNA triphosphatase were also evaluated for the first time. Our thermodynamic analyses indicate that the initial association of magnesium with the cvRTPase is dominated by a favorable entropic effect and is accompanied by the release of eight water molecules from the enzyme. Moreover, both fluorescence spectroscopy and circular dichroism assays indicated that minor conformational changes were occurring upon magnesium binding. Mutational studies were also performed and confirmed the importance of three specific glutamate residues located in the active site of the enzyme for the binding of magnesium ions. Finally, in contrast to the yeast RNA triphosphatase, we demonstrate that the binding of magnesium ions to the cvRTPase does not lead to the stabilization of the ground state binding of the RNA substrate. Based on the results of the present study, we hypothesize that the binding of magnesium ions induces local conformational perturbations in the active site residues that ultimately positions the lateral chains of critical amino acids involved in catalysis. Our results highlight fundamental differences in the role of magnesium ions in the phosphohydrolase reactions catalyzed by the cvRTPase and the closely related yeast RNA triphosphatase.
Département de Biochimie, RNA Group, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
Marie F
MF
Soulière
Jean-Pierre
JP
Perreault
Martin
M
Bisaillon
D017766:Acid Anhydride Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D002942:Circular Dichroism; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D008274:Magnesium / Q000737:chemistry*; D017862:Phycodnaviridae / Q000201:enzymology*; D011485:Protein Binding; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D014764:Viral Proteins; D017766:Acid Anhydride Hydrolases; C023090:RNA triphosphatase; D008274:Magnesium