X-ray, neutron and NMR studies of the catalytic mechanism of aspartic proteinases

Abstract

Current proposals for the catalytic mechanism of aspartic proteinases are largely based on X-ray structures of bound oligopeptide inhibitors possessing non-hydrolysable analogues of the scissile peptide bond. Until recent years, the positions of protons on the catalytic aspartates and the ligand in these complexes had not been determined with certainty due to the inadequate resolution of these analyses. There has been much interest in locating the catalytic protons at the active site of aspartic proteinases since this has major implications for detailed understanding of the mechanism of action and the design of improved transition state mimics for therapeutic applications. In this review we discuss the results of studies which have shed light on the locations of protons at the catalytic centre. The first direct determination of the proton positions stemmed from neutron diffraction data collected from crystals of the fungal aspartic proteinase endothiapepsin bound to a transition state analogue (H261). The neutron structure of the complex at a resolution of 2.1 A provided evidence that Asp 215 is protonated and that Asp 32 is the negatively charged residue in the transition state complex. Atomic resolution X-ray studies of inhibitor complexes have corroborated this finding. A similar study of the native enzyme established that it, unexpectedly, has a dipeptide bound at the catalytic site which is consistent with classical reports of inhibition by short peptides and the ability of pepsins to catalyse transpeptidation reactions. Studies by NMR have confirmed the findings of low-barrier and single-well hydrogen bonds in the complexes with transition state analogues.

Neutron crystallography: opportunities, challenges, and limitations

“…[11] have determined the H-bond arrangement in the tetrahedral binding motif formed by these aspartates and a difluoroketone ( gem -diol) inhibitor (Figure 1a). This complex is a more complete analog of the transition state than a previous one with a hydroxyethylene inhibitor [12], and provides a snapshot of possible proton transfer during the reaction.…”

Dynamic and Electrostatic Effects on the Reaction Catalyzed by HIV-1 Protease

“…16 The relatively simple structure of the active site is nevertheless the origin of some problems, which are still the source of many scientific debates. Generally, experimental 17, 18, 19, 20 and theoretical 12, 13, 21, 22, 23, 24 studies have provided evidence that peptide bond breaking is the result of a mechanism where a water molecule is activated by an aspartate residue and it then attacks as a nucleophile on a carbonyl carbon of the substrate peptide chain. Nevertheless, in the past 30 years different mechanisms for the reaction catalyzed by aspartic proteases have been suggested.…”

NMR Study of the Inhibition of Pepsin by Glyoxal Inhibitors: Mechanism of Tetrahedral Intermediate Stabilization by the Aspartyl Proteases

“…nificantly different from those of earlier studies on -secretases (16) and endathiapepsin (17,18). We discuss the role of hydrogen bonding in stabilizing gem-diols formed during catalysis and on binding inhibitors.…”