Thermodynamics of ligand binding by the yeast mRNA-capping enzyme reveals different modes of binding.
RNA-capping enzymes are involved in the synthesis of the cap structure found at the 5'-end of eukaryotic mRNAs. The present study reports a detailed study on the thermodynamic parameters involved in the interaction of an RNA-capping enzyme with its ligands. Analysis of the interaction of the Saccharomyces cerevisiae RNA-capping enzyme (Ceg1) with GTP, RNA and manganese ions revealed significant differences between the binding forces that drive the interaction of the enzyme with its RNA and GTP substrates. Our thermodynamic analyses indicate that the initial association of GTP with the Ceg1 protein is driven by a favourable enthalpy change (DeltaH=-80.9 kJ/mol), but is also clearly associated with an unfavourable entropy change (TDeltaS=-62.9 kJ/mol). However, the interaction between Ceg1 and RNA revealed a completely different mode of binding, where binding to RNA is clearly dominated by a favourable entropic effect (TDeltaS=20.5 kJ/mol), with a minor contribution from a favourable enthalpy change (DeltaH=-5.3 kJ/mol). Fluorescence spectroscopy also allowed us to evaluate the initial binding of GTP to such an enzyme, thereby separating the GTP binding step from the concomitant metal-dependent hydrolysis of GTP that results in the formation of a covalent GMP-protein intermediate. In addition to the determination of the energetics of ligand binding, our study leads to a better understanding of the molecular basis of substrate recognition by RNA-capping enzymes.
Département de biochimie, Faculté de médecine, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4.
Isabelle
I
Bougie
Amélie
A
Parent
Martin
M
Bisaillon
D002412:Cations / Q000378:metabolism; D002942:Circular Dichroism / Q000379:methods; D005453:Fluorescence; D006160:Guanosine Triphosphate / Q000378:metabolism; D008024:Ligands; D008670:Metals / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D012331:RNA, Fungal / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity; D013816:Thermodynamics; D000098556:mRNA Guanylyltransferases
D002412:Cations; D008024:Ligands; D008670:Metals; D012331:RNA, Fungal; D006160:Guanosine Triphosphate; D009713:Nucleotidyltransferases; D000098556:mRNA Guanylyltransferases