A dianionic phosphorane intermediate and transition states in an associative A(N)+D(N) mechanism for the ribonucleaseA hydrolysis reaction.

The RNaseA enzyme efficiently cleaves phosphodiester bonds in the RNA backbone. Phosphoryl transfer plays a central role in many biochemical reactions, and this is one of the most studied enzymes. However, there remains considerable controversy about the reaction mechanism. Most of this debate centers around the roles of the conserved residues, structures of the transition state or states, the possibility of a stable intermediate, and the charge and structure of this intermediate. In this communication we report calculations of the mechanism of the hydrolysis step in this reaction using a comprehensive QM/MM theoretical approach that includes a high level calculation of the interactions in the QM region, free energy estimates along an NEB optimized reaction path, and the inclusion of the interaction of the protein surroundings and solvent. Contrary to prior calculations we find a stable pentacoordinated dianionic phosphorane intermediate in the reaction path supporting an A(N)+D(N) reaction mechanism. In the transition state in the path from the reactant to the intermediate state (with barrier of 3.96 kcal/mol and intermediate stability of 2.21 kcal/mol) a proton from the attacking water is partially transferred to the His119 residue and the PO bond only partially formed from the remaining nucleophilic OH(-) species (bond order (BO) 0.11). In passing from the intermediate to the product state (barrier 13.22 kcal/mol) the PO bond on the cyclic phosphorane intermediate is nearly broken (BO 0.28) and the transfer of the proton from the Lys41 is almost complete (Lys41-H BO 0.87). In the product state a proton has been transferred from Lys41 to the O2' position of the sugar. The role of Lys41 as the catalytic acid is a result of the relative positioning of the Lys41 and His12 in the catalytic site. This configuration is supported by calculations and docking studies.