Coupling of the Guanosine Glycosidic Bond Conformation and the Ribonucleotide Cleavage Reaction: Implications for Barnase Catalysis

To examine the possible relationship of guanine-dependent GpA conformations with ribonucleotide cleavage, two potential of mean force (PMF) calculations were performed in aqueous solution. In the first calculation, the guanosine glycosidic (Gχ) angle was used as the reaction coordinate, and computations were performed on two GpA ionic species: protonated (neutral) or deprotonated (negatively charged) guanosine ribose O2’. Similar energetic profiles were obtained for both ionic forms, with two minima (anti and syn Gχ). In both simulations the anti conformation was more stable than the syn, and barriers of ~4 kcal/mol for the anti → syn transition were obtained. Structural analysis showed a remarkable sensitivity of the phosphate moiety to Gχ rotation, suggesting a possible connection between Gχ orientation and the mechanism of ribonucleotide cleavage. This hypothesis was confirmed by the second PMF calculations, for which the O2’-P distance for the deprotonated GpA was used as reaction coordinate. The computations were performed from two selected starting points: the anti and syn minima determined in the first PMF study of the deprotonated guanosine ribose O2’. The simulations revealed that the O2’ attack along the syn Gχ was more favorable than that along the anti Gχ: energetically, significantly lower barriers were obtained in the syn than in the anti conformation for the O-P bond formation; structurally, a lesser O2’-P initial distance and a better suited orientation for an in-line attack was observed in the syn relative to the anti conformation. These results are consistent with the barnase-ribonucleotide catalytic interaction, for which a guanine syn conformation of the substrate is required to allow the abstraction of the ribose H2’ proton by the general base Glu73, thereby suggesting a coupling between reactive substrate conformation and enzyme structure and mechanis