A neutron laue diffraction study of endothiapepsin: Implications for the aspartic proteinase mechanism

Current proposals for the catalytic mechanism of aspartic proteinases are l argely based on X-ray structures of bound oligopeptide inhibitors possessin g nonhydrolyzable analogues of the scissile peptide bond. However, the posi tions of protons on the catalytic aspartates and the ligand in these comple xes have not been determined with certainty. Thus, our objective was to loc ate crucial protons at the active site of an inhibitor complex since this w ill have major implications for a detailed understanding of the mechanism o f action. We have demonstrated that high-resolution neutron diffraction dat a can be collected from crystals of the fungal aspartic proteinase endothia pepsin bound to a transition state analogue (H261). The neutron structure o f the complex has been refined at a resolution of 2.1 Angstrom to an R-fact or of 23.5% and an R-free of 27.4%. This work represents the largest protei n structure studied to date by neutron crystallography at high resolution. The neutron data demonstrate that 49% of the main chain nitrogens have exch anged their hydrogen atoms with D2O in the mother liquor. The majority of r esidues resisting exchange are buried within core beta -sheet regions of th e molecule. The neutron maps confirm that the protein has a number of burie d ionized carboxylate groups which are likely to give the molecule a net ne gative charge even at very low pH, thereby accounting for its low pI. The f unctional groups at the catalytic center have clearly undergone H-D exchang e despite being buried by the inhibitor occupying the active site cleft. Mo st importantly, the data provide convincing evidence that Asp 215 is proton ated and that Asp 32 is the negatively charged residue in the transition st ate complex. This has an important bearing on mechanistic proposals for thi s class of proteinase.