TRANSPHOSPHORYLATION CATALYZED BY RIBONUCLEASE-A - COMPUTATIONAL STUDY USING AB-INITIO EFFECTIVE FRAGMENT POTENTIALS

The transphosphorylation step in the enzyme-catalyzed hydrolysis of ph osphate eaters by Ribonuclease A (RNase A) is explored using ab initio quantum chemical methods. For the first time, components found in the RNase A active site are included in the all-electron chemical model, made up of 2-hydroxyethyl methyl phosphate monoanion used as the subst rate, and small model compounds used to mimic the three important resi dues, His-12, His-119, and Lys-41, found in the RNase A active site. T he remainder of the immediate active site, including ten residues and six bound water molecules, is treated using effective fragment potenti als (EFPs) incorporated directly into the Hamiltonian of the quantum s ystem. The EFPs, derived from separate quantum calculations on individ ual components, are constructed to accurately represent the correct el ectrostatics and polarization fields of each component. High-resolutio n X-ray crystallographic data are used to assign the fixed relative po sitions of each component in the quantum and EFP regions. Characteriza tion of the salient stationary points along the transphosphorylation r eaction pathway at the RHF level using a 3-21+G(d) basis set reveals s everal low-barrier proton transfer steps between the substrate and the active site residues which allow transphosphorylation to occur with m odest activation, consistent with the experimental data. Moller-Plesse t perturbation theory (MP2) and density functional theory methods util izing a larger 6-31+G(d) basis are also used to explore the effects of electron correlation on the surface energetics. Consistent with expec tations, the electrostatic field effects from the EFPs used to represe nt the non-participating parts of the active site are found to differe ntially stabilize certain structures along the reaction pathway.