Molecular dynamics simulation of bovine pancreatic ribonuclease A-CpA and transition state-like complexes

Abstract

The mechanisms of enzymes are intimately connected with their overall structure and dynamics in solution. Experimentally, it is considerably challenging to provide detailed atomic level information about the conformational events that occur at different stages along the chemical reaction path. Here, theoretical tools may offer new potential insights that complement those obtained from experiments that may not yield an unambiguous mechanistic interpretation. In this study, we apply molecular dynamics simulations of bovine pancreatic ribonuclease A, an archetype ribonuclease, to study the conformational dynamics, structural relaxation, and differential solvation that occur at discrete stages of the transesterification and cleavage reaction. Simulations were performed with explicit solvation with rigorous electrostatics and utilize recently developed molecular mechanical force field parameters for transphosphorylation and hydrolysis transition state analogues. Herein, we present results for the enzyme complexed with the dinucleotide substrate cytidilyl-3',5'-adenosine (CpA) in the reactant, and transphosphorylation and hydrolysis transition states. A detailed analysis of active site structures and hydrogen-bond patterns is presented and compared. The integrity of the overall backbone structure is preserved in the simulations and supports a mechanism whereby His12 stabilizes accumulating negative charge at the transition states through hydrogen-bond donation to the nonbridge oxygens. Lys41 is shown to be highly versatile along the reaction coordinate and can aid in the stabilization of the dianionic transition state, while being poised to act as a general acid catalyst in the hydrolysis step.

FIG. 1

Structure of the Reactant model at top, [Trans]^TS in the middle and [Hyd]^TS at the bottom. Some MD average distances between heavy atoms are depicted.

FIG. 2

Distribution of water around phosphorus atom, phosphate and phosphorane oxygens's atoms is shown for reactant model in black, [Trans]^TS model in red and [Hyd]^TS model in green. O_2′ is at top left corner, at top right corner O_5′/O_3T is depicted, phosphorus atom is in the middle left and on the right side is O_3′ oxygen, finally O_1P is at the botton left corner and at the right corner O_2P are shown. Reactant H_2′, which is at top left corner, represents the reactant's 2′-hydroxyl proton's RDF.

Scheme 1

Schematic representation of the RNase A active site with a bound RNA ligand (CpA). The subsites interacting with RNA bases (B_i) and phosphate groups (P_i) are indicated for the reactant state (left). The transphosphorylation ([Trans]^TS) and hydrolysis ([Hyd]^TS) transition state mimics are shown in the middle and right, respectively. The atomic names and torsional angles discussed in the text are indicated.