The structure of mucor pusillus pepsin (EC 3.4.23.6), the aspartic proteinase from Mucor pusillus, has been refined to a crystallographic R -factor of 16·2% at 2·0 Å resolution. The positions of 2638 protein atoms, 221 solvent atoms and a sulphate ion have been determined with an estimated root-mean-square (r.m.s.) error of 0·15 to 0·20 Å. In the final model, the r.m.s. deviation from ideality for bond distances is 0·022 Å, and for angle distances it is 0·050 Å. Comparison of the overall three-dimensional structure with other aspartic proteinases shows that mucor pusillus pepsin is as distant from the other fungal enzymes as it is from those of mammalian origin. Analysis of a rigid body shift of residues 190 to 302 shows that mucor pusillus pepsin displays one of the largest shifts relative to other aspartic proteinases (14·4° relative to endothiapepsin) and that changes have occurred at the interface between the two rigid bodies to accommodate this large shift. A new sequence alignment has been obtained on the basis of the three-dimensional structure, enabling the positions of large insertions to be identified. Analysis of secondary structure shows the β-sheet to be well conserved whereas α-helical elements are more variable. A new α-helix hN4 is formed by a six-residue insertion between positions 131 and 132. Most insertions occur in loop regions: -5 to 1 (five residues relative to porcine pepsin); 115 to 116 (six residues); 186 to 187 (four residues); 263 to 264 (seven residues); 278 to 279 (four residues); and 326 to 332 (six residues). The active site residues are highly conserved in mucor pusillus pepsin; r.m.s. difference with rhizopuspepsin is 0·37 Å for 25 Cα atom pairs. However, residue 303, which is generally conserved as an aspartate, is changed to an asparagine in mucor pusillus pepsin, possibly influencing pH optimum. Substantial changes have occurred in the substrate binding cleft in the region of S1 and S3 due to the insertion between 115 and 116 and the rearrangement of loop 9-13. Residue Asn219 necessitates a shift in position of substrate main-chain atoms to maintain hydrogen bonding pattern. Invariant residues Asp11 and Tyr14 have undergone a major change in conformation apparently due to localized changes in molecular structure. Both these residues have been implicated in zymogen stability and activation.

To aid in the design of an effective inhibitor to human renin, it is essential to have a detailed knowledge of how this aspartic proteinase interacts with its substrate, angiotensinogen. Human renin shows a stringent specificity toward the Leu-Val bond in its natural substrate. The minimal length for an effective substrate has been characterised as an octapeptide sequence derived from the amino terminal portion of angiotensinogen (residues 6----13): His-Pro-Phe-His-Leu-Val-Ile-His (Leu-Val is the scissile bond). This suggests that renin has a fairly extensive active site cleft, as has been observed in homologous enzymes whose three-dimensional structures have been solved using x-ray diffraction methods. The homologous fungal aspartic proteinase, endothiapepsin, has been cocrystallised with human renin inhibitors of the type His-Pro-Phe-His-Leu-R-Val-Ile-His, where R indicates a reduced carbonyl analogue of the scissile peptide bond. The three-dimensional crystallographic structures of two complexes of endothiapepsin with an inhibitor have been solved. The details of inhibitor binding at the active site cleft of endothiapepsin are described. These data allow a rational approach to the design of novel renin inhibitors, through studies of these inhibitors in a three-dimensional model of human renin constructed in our laboratory.

Conference Article| August 01 1987 Inhibitors of aspartic proteinases and their relevance to the design of antihypertensive agents J. B. COOPER; J. B. COOPER *Laboratory of Molecular Biology, Department of Crystallography, Birkbeck College, University of London, Malet Street, London WCIE 7HX. U.K. Search for other works by this author on: This Site PubMed Google Scholar S. I. FOUNDLING; S. I. FOUNDLING *Laboratory of Molecular Biology, Department of Crystallography, Birkbeck College, University of London, Malet Street, London WCIE 7HX. U.K. Search for other works by this author on: This Site PubMed Google Scholar A. HEMMINGS; A. HEMMINGS *Laboratory of Molecular Biology, Department of Crystallography, Birkbeck College, University of London, Malet Street, London WCIE 7HX. U.K. Search for other works by this author on: This Site PubMed Google Scholar F. E. WATSON; F. E. WATSON *Laboratory of Molecular Biology, Department of Crystallography, Birkbeck College, University of London, Malet Street, London WCIE 7HX. U.K. Search for other works by this author on: This Site PubMed Google Scholar B. L. SIBANDA; B. L. SIBANDA *Laboratory of Molecular Biology, Department of Crystallography, Birkbeck College, University of London, Malet Street, London WCIE 7HX. U.K. Search for other works by this author on: This Site PubMed Google Scholar T. L. BLUNDELL; T. L. BLUNDELL *Laboratory of Molecular Biology, Department of Crystallography, Birkbeck College, University of London, Malet Street, London WCIE 7HX. U.K. Search for other works by this author on: This Site PubMed Google Scholar D. M. JONES; D. M. JONES †Department of Chemical Pathology, Royal Postgraduate Medical School, London W12 OHS, U.K. Search for other works by this author on: This Site PubMed Google Scholar A. HALLETT; A. HALLETT †Department of Chemical Pathology, Royal Postgraduate Medical School, London W12 OHS, U.K. Search for other works by this author on: This Site PubMed Google Scholar B. ATRASH; B. ATRASH †Department of Chemical Pathology, Royal Postgraduate Medical School, London W12 OHS, U.K. Search for other works by this author on: This Site PubMed Google Scholar M. SZELKE M. SZELKE †Department of Chemical Pathology, Royal Postgraduate Medical School, London W12 OHS, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1987) 15 (4): 751–754. https://doi.org/10.1042/bst0150751 Article history Received: February 23 1987 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation J. B. COOPER, S. I. FOUNDLING, A. HEMMINGS, F. E. WATSON, B. L. SIBANDA, T. L. BLUNDELL, D. M. JONES, A. HALLETT, B. ATRASH, M. SZELKE; Inhibitors of aspartic proteinases and their relevance to the design of antihypertensive agents. Biochem Soc Trans 1 August 1987; 15 (4): 751–754. doi: https://doi.org/10.1042/bst0150751 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1987 Biochemical Society1987 Article PDF first page preview Close Modal You do not currently have access to this content.

Inhibitors of the conversion of angiotensinogen to the vasoconstrictor angiotensin II have considerable value as antihypertensive agents. For example, captopril and enalapril are clinically useful as inhibitors of angiotensin-converting enzyme. This has encouraged intense activity in the development of inhibitors of kidney renin, which is a very specific aspartic proteinase catalysing the first and rate limiting step in the conversion of angiotensinogen to angiotensin II. The most effective inhibitors such as H-142 and L-363,564 have used non-hydrolysable analogues of the proposed transition state, and partial sequences of angiotensinogen (Table 1). H-142 is effective in lowering blood pressure in humans but has no significant effect on other aspartic proteinases such as pepsin in the human body (Table 1). At present there are no crystal structures available for human or mouse renins although three-dimensional models demonstrate close structural similarity to other spartic proteinases. We have therefore determined by X-ray analysis the three-dimensional structures of H-142 and L-363,564 complexed with the aspartic proteinase endothiapepsin, which binds these inhibitors with affinities not greatly different from those measured against human renin (Table 1). The structures of these complexes and of that between endothiapepsin and the general aspartic proteinase inhibitor, H-256 (Table 1) define the common hydrogen bonding schemes that allow subtle differences in side-chain orientations and in the positions of the transition state analogues with respect to the active-site aspartates.