Mechanism of action of aspartic proteinases: Application of transition-state analogue theory

Summary

Applying the semiempirical MO methods AM1 and PM3 as well as the density functional theory to the model of the catalytic site composed of ca. 160–190 atoms, we have carried out studies aimed at the explanation of three aspects of the mechanism of action of aspartic proteinases: the site of dissociation within the catalytic diad COOH/COO^- (i) in the free enzyme and (ii) in the Michaelis complex, and (iii) the energy changes associated with the catalytic paths. We have found that the state of dissociation within the catalytic diad is ligand-sensitive. In the free enzyme and in the intermediate complexes, Asp³³ prefers to be dissociated with the outer oxygen of Asp²¹³ protonated, while in the Michaelis and product complexes the opposite holds true. This is in agreement with recent mechanistic hypotheses and with some experimental results by FTIR and NMR. The energy diagram for the catalysis indicates that electronic effects are responsible most of all for the relative reduction of energy of the intermediates and possibly transition states on the catalytic reaction path. The shape of the diagram qualitatively agrees with the transition-state analogue theory for the enzymatic reactions.