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

School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton, SO16 7PX, England.
European Biophysics Journal (Impact Factor: 2.22). 10/2006; 35(7):559-66. DOI: 10.1007/s00249-006-0065-7
Source: PubMed


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.

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    • "That is, in the crystal structure, the two Asp dyad protonation states shown schematically in (Fig. 6) are equally populated. Overall, the work described above has provided the first structural evidence that low-barrier hydrogen bonds may be significant in the catalytically driven reaction pathway in aspartic proteases and has helped to locate the LBHB interactions in the catalytic centre , thus giving a sounder footing for evaluating their role in the mechanism [30]. "
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