An ab initio study of the first stage of catalysis in the monomeric aspartic proteinases

Abstract

We have studied the initial stages of hydrolysis in the monomeric aspartic proteinases by performing ab initio calculations (Hartree–Fock, MP2, DFT) on the active site of endothiapepsin. The crystallographic coordinates of a difluorostatone inhibitor complexed with endothiapepsin were used to generate a template for the initial enzyme/substrate complex. The active site was modelled as a formic acid–formate anion moiety (representing the catalytic aspartates, Asp-32 and Asp-215) and a bound water molecule. Acetamide was used to represent the substrate, and residues Gly-34, Ser-35, Gly-217, and Thr-218, which all interact with the active site, were modelled using formamide and methanol. The calculations predict that T218 plays a direct role in catalysis by forming a strong hydrogen bond with the water oxygen during the initial stages of nucleophilic attack. S35 plays an indirect role by lowering the p K a of Asp-32 and facilitating the formation of the gem–diol intermediate. The predicted reaction pathway is unusual because it involves two local minima, with very short O water...C substrate distances, which are stabilised by T218.