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

Abstract

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.

Molecular Dynamics and Free Energy Analyses of Cathepsin D−Inhibitor Interactions: Insight into Structure-Based Ligand Design

“…Because the binding interface of CatD and its ligand is similar to that of plasmepsin II-pepstatin, we protonated His77 and Glu260 . The electronic structure calculations on the penicillopepsin−substrate complex suggested that the Asp33 outer oxygen was protonated . Because of the high structural similarity at the active site among the aspartic proteinases, we chose the Asp33 outer oxygen to be protonated in CatD−inhibitor complexes.…”

“…38 The electronic structure calculations on the penicillopepsin-substrate complex suggested that the Asp33 outer oxygen was protonated. 39 Because of the high structural similarity at the active site among the aspartic proteinases, we chose the Asp33 outer oxygen to be protonated in CatD-inhibitor complexes. Seven ligands were chosen from the combinatorial libraries of CatD inhibitors with the K i values ranging from nanomolar to micromolar.…”

Macrocyclic Inhibitors of Penicillopepsin. 2. X-ray Crystallographic Analyses of Penicillopepsin Complexed with a P3−P1 Macrocyclic Peptidyl Inhibitor and with Its Two Acyclic Analogues

“…Subsequently, similar characteristics were reported in a crystallographic analysis of a complex between the human immunodeficiency virus type 1 (HIV-1) proteinase and its competitive inhibitor, a C 2 -symmetric phosphinate . This working model of the aspartyl proteinases complexed with transition-state analogues has recently received more support from crystal structure analyses such as those of pepsin, rhizopuspepsin, and endothiapepsin and from Fourier transform infrared spectroscopic studies. , In addition, an energetic analysis for such a catalytic pathway suggested that electronic effects are mainly responsible for the relative reduction of energy of the intermediates and possibly transition states on the catalytic reaction path …”