Investigation on the reaction mechanism of ribonuclease enzymes

This is the authors' abstract. We don't add key points for this paper.

Pancreatic ribonuclease A (RNase A) is an efficientenzyme for the hydrolysis of phosphate-ester linkages insingle-stranded RNA. There is strong evidence that the cat-alytic process occurs via a two-step general acid-basemechanism. The first step involves intramoleculartransphosphorylation leading to a trigonal bipyramidalcyclic phosphorane intermediate and displacement of theO5'-nucleotide product. In the subsequent step, the cyclicphosphorane is hydrolyzed to a 3'-monophosphatenucleotide as the final product. Three amino acid residueswithin the active site, Lys41, His12 and His119, areknown to play significant roles in the catalytic process(Figure 1). They are believed to act as a general base andgeneral acid. Because of the availability of high resolutionX-ray and neutron scattering structural data [4], RNase Aattracts considerable attention as a target for theoreticalstudies [1][2][3][5][6].The purpose of this research program was to investigatethe reaction mechanism of this enzyme using high levelquantum chemistry methods of NWChem [7] with thehighest feasible accuracy (DFT/B3LYP, Ahlrichs-pVDZbasis set) to provide an accurate representation of thechemistry of the active site (QM region) and molecularmechanics (Amber 99) to describe the remaining proteinand solvent (MM region).For this enzyme reaction there is no data providing adetailed picture of any structures along the reaction path[5]. However, there are structural data for a vanadate com-plex [4], which has been accepted as a transition state ana-logue. After evaluation of our computational analysis wesubstituted the central vanadium by phosphorus to modelthe intermediate state.Afterwards we elongated the endocyclic and exocyclic P-Obonds to find reactant and product states allowing forrelaxation at each step. In addition a thermally averagedNEB method has been implemented to produce an unbi-ased view of the movement of atoms as the system evolvesalong the reaction path. Free energy calculations havebeen performed to account for thermal fluctuations of theprotein environment. This way we could calculate theenergy profile of the hydrolysis step and identify the tran-sition state structures along the reaction coordinate.