The structure of a synthetic pepsin inhibitor complexed with endothiapepsin

Abstract

The conformation of a synthetic polypeptide inhibitor, bound to the active site of the fungal aspartic proteinase endothiapepsin (EC 3.4.23.6), has been determined by X-ray diffraction at 0.20-nm resolution and refined to an agreement factor of 0.20. The inhibitor:is based on a chromogenic substrate of pepsin (EC 3.4.23.1). It has, in place of the scissile bond, a reduced peptide group which is resistant to hydrolysis and mimics the tetrahedral transition state. The inhibitor binds in an extended conformation with the reduced bond close to the essential aspartate side-chains of the enzyme. The hydrogen bonds and hydrophobic interactions between the enzyme and the inhibitor do not induce large conformational changes.The aspartic proteinases, a family of enzymes including pepsin, are characterized by two essential aspartate residues (32 and 215 in pepsin) which have been shown by X-ray diffraction to reside at the active site [l -41. Pepsin is the major digestive enzyme of the stomach and like most aspartic proteinases is optimally active at low pH. Analysis of the kinetic parameters of hydrolysis, by pepsin, of a single peptide bond in small synthetic substrates has shown that the rate constants depend not only on the nature of the residues adjacent to the scissile bond, but also on several residues each side of the dipeptidyl unit indicating that aspartic proteinases have up to seven specificity subsites [5, 61. A study of the specificity of porcine pepsin [7] for tripeptides of the type : benzyloxycarbonyl-His-Xaa-XaaOMe showed that the highest k,,,lK, is obtained when both Xaa residues are phenylalanines. Varying the Xaas causes k,,, to change much more than K , indicating that tight binding at these subsites lowers the activation barrier of hydrolysis by stabilization of the transition state. A survey of the amino acids found to occur in the vicinity of pepsin-susceptible bonds in proteins [8] led to the design and synthesis of a useful chromogenic substrate nicknamed 'Ralph' based on a PhePhe dipeptide [9], Tables 1, 2.The substrate has been converted to a potent pepsin inhibitor by replacing the scissile bond with a reduced peptide group -CH2-NH-giving H-256 [4, 101 (Tables 1, 2) which inhibits endothiapepsin and porcine pepsin with Ki values of 60 nM and 40 nM respectively.

Side-chain Specificities and Molecular Modelling of Peptide Determinants for Two Anti-Sm B/B′ Autoantibodies

“…Side chain involvement in definition of secondary structure is implicit in the simulations. Involvement of a preferred backbone in ligand binding has been demonstrated by structures of pepsin-inhibitor complexes [22][23][24]. We know of no other descriptions of antigen-antibody interactions beyond that presented herein which suggest backbone interaction in the binding of antibody to antigen.…”

Structure of a secreted aspartic protease from C. albicans complexed with a potent inhibitor: Implications for the design of antifungal agents

“…Upper diagonal lists the results obtained from the overall superposition. lPSA, pepsin/A62095 complex (Chen et al, 1992); ISMR, mouse submaxillary gland renin (Dealwis et al, 1994); SAPZX, secreted aspartic protease from clinical isolate (this work); IEED, Endothiupurusiticu/ inhibitor complex (Cooper et al, 1987); 3APR, Rhizopushhibitor complex (Gilliland et al, 1990).…”

Comparative modelling of barley‐grain aspartic proteinase: A structural rationale for observed hydrolytic specificity

“…The three-dimensional structure of endothiapepsin complexed with the pepsin inhibitor H-256 [28] was used as a guide to model the transition state of the substrate Lys-Pro-Be-Glu-Phe-Phe(4-NO,)-ArgLeu, into the active site of these enzymes. H-256 was chosen because it contains a Pro at P,, Glu at P,, Phe at P, and Pi, and Arg at Pi, as in the substrate used.…”