Theoretical studies on the hydrolysis of phosphate diesters in the gas phase, solution, and RNase A

Density functional theory, polarizable continuum models and semiempirical h ybrid quantum mechanical/molecular mechanical (QM/MM) calculations were app lied to the hydrolysis of phosphate diesters in the gas phase, in solution, and in the enzyme RNase A. Neutralization of the negative charge of the pe ntacovalent phosphorane intermediates provides a substantial stabilization of the transition-state structures in the gas phase. Inclusion of solvent e ffects on the phosphate/phosphorane species was critical to reproducing the trends in reactivity observed experimentally. Finally, the catalytic mecha nism for the hydrolysis of uridine 2 ' ,3 ' -cyclic phosphate by RNase A wa s studied by QM/MM calculations. Our results suggest that the rate-limiting transition state of the reaction corresponds to the approach of a water mo lecule to the phosphate and its activation by His119. Thus, His119 acts as a generalized base for the reaction. The water attack leads to a pentacoval ent phosphorane transition state of formal charge -2; this excess of negati ve charge in the transition state is stabilized by a number of positively c harged residues including His12 and Lys41. In the second stage of the react ion, the phosphorane is converted into products. This part of the reaction proceeds without a detectable barrier, and it is facilitated by a proton tr ansfer from Lys41 to the departing O-2 '. (C) 2002 John Wiley & Sons, Inc.