Aspartic proteinases are an important family of enzymes associated with several pathological conditions such as hypertension (renin), gastric ulcers (pepsin), neoplastic disease (cathepsins D and E) and AIDS (HIV proteinase). These enzymes depend on two invariant Asp-Thr-Gly sequences for catalytic activity. These residues lie at the centre of an extended substrate binding cleft and are thought to polarise a bound water molecule which hydrolyses the scissile bond of the substrate. We are comparing the binding of peptide inhibitors to different enzymes to understand the basis of their specificities.
We have determined the X-ray structures at high resolution of more than 20 transition-state analogue inhibitors bound to endothiapepsin. The inhibitors adopt extended conformations in the cleft with the transition state isosteres interacting tightly with the catalytic aspartate groups. Within the last few years the structures of human renin, mouse submandibular renin, calf chymosin, porcine pepsin, mucor pepsin and yeast proteinase A have been determined by the Birkbeck group with different inhibitors bound to each. The mammalian enzymes have more extensive loop regions covering the active site cleft thereby providing stricter substrate specificity.
The above shows a superposition of 21 transition state analogue inhibitors bound to endothiapepsin. The main chain conformations are strongly conserved in the central regions of these molecules. Pockets of low specificity allow the peptide side chains to adopt a range of conformations, most notable at P2.
The peptide inhibitors studied so far bind in extended conformations making beta-sheet hydrogen bonds with the enzyme. In some pockets notably S2, the inhibitor side chains adopt a range of conformations whereas at pockets of greater specificity such as S1 and S3, the conformation of the inhibitor's side chain is severely restricted. The strict specificity of renins at the S3' subsite derives from a rigid proline-rich loop which forms one side of the pocket. Therefore, differences in rigidity of the binding pockets, the precise positioning of secondary structure elements forming the pockets and the orientation of the two lobes of the enzyme may have significant effects on its substrate specificity.
Researchers: J.Read, N.Cronin, M.Groves, M.Williams, V.Dhanaraj, J.Cooper, S.P.Wood & T.L.Blundell.
Key reference.
X-ray analyses of peptide-inhibitor complexes define the structural basis for specificity for human and mouse renins.
V.Dhanaraj, C.DeAlwis, C.Frazao, M.Badasso, B.L.Sibanda, I.J.Tickle, J.B.Cooper, H.P.C.Driessen, M.Newman, C.Aguilar, S.P.Wood, T.L.Blundell, P.M.Hobart, K.F., Geohegan, M.J.Ammirati, D.E.Danley, B.A.O'Connor, D.J.Hoover. Nature (1992) 357, 466-472.