Computational studies on aspartic proteases. I. Active‐site protonation and hydration in the substrate‐free crystalline state

Abstract

AbstractWe performed semiempirical molecular orbital calculations on the free and hydrated states of the HCOO−…HCOOH couple in crystals of an aspartic protease, rhizopuspepsin, in order to gain information not available by conventional X‐ray diffraction studies. The reliability of the MNDO/PM3 method was proven for the model system HCOO−…HOH, for which we could reproduce geometry and energetics of the complex, obtained by sophisticated ab initio calculations, with astonishing accuracy. Comparing the geometries of the active‐site models to experiment and their relative energies, we suggest that in the substrate‐free crystalline state the carboxyl–carboxylate–hydroxonium triad exists most probably in the neutral form. The carboxyl–carboxylate dyad attracts a hydroxonium ion better than does ammonium, indicating that the nonhydrogen atom, located by X‐ray crystallography near the active site, is oxygen and not nitrogen. We located the most probable hydrogen positions by geometry optimization.