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 recently, 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 transition state mimics which might have therapeutic applications. It has been demonstrated that high resolution neutron diffraction data can be collected from crystals of the fungal aspartic proteinase endothiapepsin bound to a transition state analogue (H261). The neutron structure of the complex has been refined at a resolution of 2.1Å to an R-factor of 23.57% and an R-free of 27.4%. Most importantly the data provide convincing evidence that Asp 215 is protonated and that Asp 32 is the negatively charged residue in the transition state complex. In parallel, atomic resolution X-ray studies ofinhibitor complexes have corroborated this finding. Interestingly, 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. All of these results have an important bearing on mechanistic proposals for this class of proteinase.
|Title of host publication||Hydrogen- and Hydration-Sensitive Structural Biology|
|Editors||N. Nobuo, M. Hiroshi, J. R. Helliwell, E. Westhof|
|Place of Publication||Japan|
|Publisher||Kubapro Co. Ltd.|
|Number of pages||8|
|Publication status||Published - 2005|