Inhibition of aspartic proteinases by synthetic peptides derived from the propart region of human prorenin.

1. Five synthetic peptides which together spanned the propart segment of human prorenin were tested for their ability to interact with human renin, pepsin, gastricsin, cathepsin D, cathepsin E, calf chymosin and the aspartic proteinase from Endothia parasitica. 2. While two peptides showed no significant effect with any of the enzymes, a further two were cleaved by several enzymes. 3. Only one (corresponding to the 32P-43P residues in the propart sequence) acted as a weak competitive inhibitor of most of the enzymes.     

The selectivity of statine-based inhibitors against various human aspartic proteinases.

The interactions of five human enzymes (renin, pepsin, gastricsin, cathepsin D and cathepsin E) and the aspartic proteinase from Endothia parasitica with several series of synthetic inhibitors were examined. All of the inhibitors contained the dipeptide analogue statine or its phenylalanine or cyclohexylalanine homologues in the P1-P1' positions. The residues occupying the peripheral sub-sites (P4 to P3') were varied systematically and inhibitory constants were determined for the interactions with each of the proteinases. Inhibitors were elucidated that specifically inhibited human renin and did not affect any of the other human enzymes or the fungal proteinase. With suitable selection of residues to occupy individual sub-sites, effective inhibitors of specific human aspartic proteinases may now be designed.     

Molecular model for the binary complex of uropepsin and pepstatin

The three-dimensional structure of human uropepsin complexed with pepstatin has been modelled using human pepsin as a template. Uropepsin is an aspartic proteinase from the urine, produced in the form of pepsinogen A in the gastric mucosa. The structure is bilobal, consisting of two predominantly beta-sheet lobes which, as observed in other aspartic proteinases, are related by a pseudo twofold axis. A structural comparison between binary complexes of pepsin:pepstatin and uropepsin:pepstatin is discussed.     

Human renin inhibition by a diazoacyl reagent: Relationship of the enzyme to other proteinases

Human renin is inactivated by a diazoacyl compound (diazoacetylglycine ethyl ester; N₂CHCO-Gly-OEt) in the presence of Cu(II). The mechanism of the inactivation is presumably identical to that which has been determined for pepsin and several other proteinases: esterification of the β-carboxyl of an aspartic acid residue at the active site of the enzyme. Renin's inhibition by the diazoacyl reagent, its specificity toward a hydrophobic sequence, and its inhibition by pepstatin, all suggest a close relationship to the acid proteinases, especially pepsin and cathepsin D. However, renin, a neutral proteinase, would be better classified together with other diazoacyl-inhibited enzymes by active site rather than pH optimum. The term “aspartic proteinase” is suggested for this group of enzymes.     

Exploring the binding preferences/specificity in the active site of human cathepsin E

Aspartic proteinases are produced in the human body by a variety of cells. Some of these proteins, examples of which are pepsin, gastricsin, and renin, are secreted and exert their effects in the extracellular spaces. Cathepsin D and cathepsin E on the other hand are intracellular enzymes. The least characterized of the human aspartic proteinases is cathepsin E. Presented here are results of studies designed to characterize the binding specificities in the active site of human cathepsin E with comparison to othermechanistically similar enzymes. A peptide series based on Lys-Pro-Ala-Lys-Phe*Nph-Arg-Leu was generatedto elucidate the specificity in the individual binding pockets with systematic substitutions in the P_5? P₂ and P₂′-P₃′ based on charge, hydrophobicity, and hydrogen bonding. Also, to explore the S₂ binding preferences, asecond series of peptides based on Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu was generated with systematic replacements in the P₂ position. Kinetic parameters were determined forboth sets of peptides. The results were correlated to a rule-based structural model of human cathepsin E, constructed on the known three-dimensional structures of several highly homologous aspartic proteinases; porcine pepsin, bovine chymosin, yeast proteinase A, human cathepsin D, andmouse and human renin. Important specificity-determining interactions were found in the S₃ (Glu13) and S₂ (Thr-222, Gln-287, Leu-289, Ile-300)subsites. © 1995 Wiley-Liss, Inc.     

Accessing the reproducibility and specificity of pepsin and other aspartic proteases

The aspartic protease pepsin is less specific than other endoproteinases. Because aspartic proteases like pepsin are active at low pH, they are utilized in hydrogen deuterium exchange mass spectrometry (HDX MS) experiments for digestion under hydrogen exchange quench conditions. We investigated the reproducibility, both qualitatively and quantitatively, of online and offline pepsin digestion to understand the compliment of reproducible pepsin fragments that can be expected during a typical pepsin digestion. The collection of reproducible peptides was identified from > 30 replicate digestions of the same protein and it was found that the number of reproducible peptides produced during pepsin digestion becomes constant above 5–6 replicate digestions. We also investigated a new aspartic protease from the stomach of the rice field eel (Monopterus albus Zuiew) and compared digestion efficiency and specificity to porcine pepsin and aspergillopepsin. Unique cleavage specificity was found for rice field eel pepsin at arginine, asparagine, and glycine. Different peptides produced by the various proteases can enhance protein sequence coverage and improve the spatial resolution of HDX MS data. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

X-ray analyses of aspartic proteinases. IV. Structure and refinement at 2.2 A resolution of bovine chymosin

The structure of calf chymosin (EC 3.4.23.3), the aspartic proteinase from the gastric mucosa, was solved using the technique of molecular replacement. We describe the use of different search models based on distantly related fungal aspartic proteinases and investigate the effect of using only structurally conserved regions. The structure has been refined to a crystallographic R-factor of 17% at 2.2 A resolution with an estimated co-ordinate error of 0.21 A. In all, 136 water molecules have been located of which eight are internal. The structure of chymosin resembles that of pepsin and other aspartic proteinases. However, there is a considerable rearrangement of the active-site "flap" and, in particular, Tyr75 (pepsin numbering), which forms part of the specificity pockets S1 and S1'. This is probably a consequence of crystal packing. Electrostatic interactions on the edge of the substrate binding cleft appear to account for the restricted proteolysis of the natural substrate kappa-casein by chymosin. The local environment of invariant residues is examined, showing that structural constraints and side-chain hydrogen bonding can play an important role in the conservation of particular amino acids.     

Solution Structure of the Squash Aspartic Acid Proteinase Inhibitor (SQAPI) and Mutational Analysis of Pepsin Inhibition

The squash aspartic acid proteinase inhibitor (SQAPI), a proteinaceous proteinase inhibitor from squash, is an effective inhibitor of a range of aspartic proteinases. Proteinaceous aspartic proteinase inhibitors are rare in nature. The only other example in plants probably evolved from a precursor serine proteinase inhibitor. Earlier work based on sequence homology modeling suggested SQAPI evolved from an ancestral cystatin. In this work, we determined the solution structure of SQAPI using NMR and show that SQAPI shares the same fold as a plant cystatin. The structure is characterized by a four-strand anti-parallel β-sheet gripping an α-helix in an analogous manner to fingers of a hand gripping a tennis racquet. Truncation and site-specific mutagenesis revealed that the unstructured N terminus and the loop connecting β-strands 1 and 2 are important for pepsin inhibition, but the loop connecting strands 3 and 4 is not. Using ambiguous restraints based on the mutagenesis results, SQAPI was then docked computationally to pepsin. The resulting model places the N-terminal strand of SQAPI in the S′ side of the substrate binding cleft, whereas the first SQAPI loop binds on the S side of the cleft. The backbone of SQAPI does not interact with the pepsin catalytic Asp³²–Asp²¹⁵ diad, thus avoiding cleavage. The data show that SQAPI does share homologous structural elements with cystatin and appears to retain a similar protease inhibitory mechanism despite its different target. This strongly supports our hypothesis that SQAPI evolved from an ancestral cystatin.     

Revised 2.3 A structure of porcine pepsin: evidence for a flexible subdomain

A revised three-dimensional crystal structure of ethanol-inhibited porcine pepsin refined to an R-factor of 0.171 at 2.3 A resolution is presented and compared to the refined structures of the fungal aspartic proteinases: penicillopepsin, rhizopuspepsin, and endothiapepsin. Pepsin is composed of two nearly equal N and C domains related by an intra dyad. The overall polypeptide fold and active site structures are homologous for pepsin and the fungal enzymes. The weak inhibition of pepsin by ethanol can be explained by the presence of one or more ethanol molecules, in the vicinity of the active site carboxylates, which slightly alter the hydrogen-bonding network and which may compete with substrate binding in the active site. Structural superposition analysis showed that the N domains aligned better than the C-domains for pepsin and the fungal aspartic proteinases: 107-140 C alpha pairs aligned to 0.72-0.85 A rms for the N domains; 64-95 C alpha pairs aligned to 0.78-1.03 A rms for the C domains. The major structural difference between pepsin and the fungal enzymes concerns a newly described subdomain whose conformation varies markedly among these enzyme structures. The subdomain in pepsin comprises nearly 100 residues and is composed of two contiguous segments within the C domain (residues 192-212 and 223-299). the subdomain is connected, or "hinged," to a mixed beta-sheet that forms one of the structurally invariant, active site psi-loops. Relative subdomain displacements as large as a 21.0 degrees rotation and a 5.9 A translation were observed among the different enzymes. There is some suggestion in pepsin that the subdomain may be flexible and perhaps plays a structural role in mediating substrate binding, determining the substrate specificity, or in the activation of the zymogen.     

Cleavage of human big endothelin-1 by Candida albicans aspartic proteinase

Abstract A Candida albicans aspartic proteinase (CAP), one of the secretory proteinases of Candida albicans , is thought to be a possible virulence factor in Candida albicans infection. Whereas endothelin-1 is found as an endothelium-derived strong vasoconstrictive peptide, it is known to have a role in the maintenance of vascular homeostasis and tissue survival. Endothelin-1 is generated from a precursor form of endothelin-1, the so-called big endothelin-1. It has recently been reported that cathepsin D, E and pepsin, which are aspartic proteinases, convert big endothelin-1 to endothelin-1. In this study, the relationship between CAP and big endothelin-1 was studied. High performance liquid chromatography analysis revealed that big endothelin-1 was cleaved into several amino acid sites by CAP, but endothelin-1 was not converted from big endothelin-1. CAP cleaved big endothelin-1 at different sites when compared with that of other known aspartic proteinases, and it suppressed endothelin-1 production through the degradation of big endothelin-1. CAP may break homeostatic mechanism of endothelin-1 in Candida albicans infectious lesion.     

Plant aspartic proteinases: enzymes on the way to a function

Plant aspartic proteinases have been characterized from seeds, flowers and leaves of a number of different species. The enzymes are generally either monomeric or heterodimeric, containing two peptides processed from the same precursor protein. The plant enzymes, like their mammalian and microbial counterparts, are active at acidic pH and inhibited by a class specific inhibitor pepstatin A. Plant aspartic proteinases are generally either secreted or targeted to the vacuolar/protein storage body compartment. The primary sequences of many of these enzymes have been determined and are very homologous with each other as well as with enzymes from mammalian and microbial origins. Plant aspartic proteinases, however, have a very unique plant specific region, which is not found in mammalian, microbial, or viral aspartic proteinases. The function of this region has not been elucidated. A role for these plant enzymes in protein processing or degradation has been proposed, however, more studies are required to confirm their in vivo functions. Recent intriguing results suggest possible roles for these enzymes in programmed cell-death of tissues and in pathogen resistance.     

Oestrogen-induced expression of a novel liver-specific aspartic proteinase in Danio rerio (zebrafish)

Aspartic proteinases are a group of endoproteolytic proteinases active at acidic pH and characterized by the presence of two aspartyl residues in the active site. They include related paralogous proteins such as cathepsin D, cathepsin E and pepsin. Although extensively investigated in mammals, aspartic proteinases have been less studied in other vertebrates. In a previous work, we cloned and sequenced a DNA complementary to RNA encoding an enzyme present in zebrafish liver. The sequence resulted to be homologous to a novel form of aspartic proteinase firstly described by us in Antarctic fish. In zebrafish, the gene encoding this enzyme is expressed only in the female liver, in contrast with cathepsin D that is expressed in all the tissues examined independently of the sex. For this reason we have termed the new enzyme liver-specific aspartic proteinase (LAP).Northern blot analyses indicate that LAP gene expression is under hormonal control. Indeed, in oestrogen-treated male fish, cathepsin D expression was not enhanced in the various tissues examined, but the LAP gene product appeared exclusively in the liver. Our results provide evidence for an oestrogen-induced expression of LAP gene in liver. We postulate that the sexual dimorphic expression of the LAP gene may be related to the reproductive process.     

A history of pepsin and related enzymes

Studies on gastric digestion during 1820-1840 led to the discovery of pepsin as the agent which, in the presence of stomach acid, causes the dissolution of nutrients such as meat or coagulated egg white. Soon afterward it was shown that these protein nutrients were cleaved by pepsin to diffusible products named peptones. Efforts to isolate and purify pepsin were spurred by its widespread adoption for the treatment of digestive disorders, and highly active preparations were available by the end of the nineteenth century. There was uncertainty, however, as to the chemical nature of pepsin, for some preparations exhibited the properties of proteins while other preparations failed to do so. The question was not settled until after 1930, when Northrop crystallized swine pepsin and provided convincing evidence for its identity as a protein. The availability of this purified pepsin during the 1930s also led to the discovery of the first synthetic peptide substrates for pepsin, thus providing needed evidence for the peptide structure of native proteins, a matter of debate at that time. After 1945, with the introduction of new separation methods, notably chromatography and electrophoresis, and the availability of specific proteinases, the amino acid sequences of many proteins, including pepsin and its precursor pepsinogen, were determined. Moreover, treatment of pepsin with chemical reagents indicated the participation in the catalytic mechanism of two aspartyl units widely separated in the linear sequence. Studies on the kinetics of pepsin action on long chain synthetic peptides suggested that the catalytic site was an extended structure. Similar properties were found for other "aspartyl proteinases," such as chymosin (used in cheese making), some intracellular proteinases (cathepsins), and plant proteinases. After 1975, the three-dimensional structures of pepsin and many of its relatives were determined by means of x-ray diffraction techniques, greatly extending our insight into the mechanism of the catalytic action of these enzymes. That knowledge has led to the design of new inhibitors of aspartyl proteinases, which are participants in the maturation of human immunodeficiency virus and in the generation of Alzheimer's disease.     

Slow step after bond-breaking by porcine pepsin identified using solvent deuterium isotope effects

The relatively fast artificial substrate Leu-Ser-rho-nitro-Phe-Nle-Ala-Leu-OMe generates a solvent isotope effect of 1.51 +/- 0.02 only on the maximal velocity of peptide hydrolysis catalyzed by porcine pepsin (EC 3.4.23.1). The absence of an isotope effect on V/K places the isotopically-sensitive step after peptide bond cleavage and the release of the first product. Reprotonation of the active site aspartic carboxyls is proposed as the most likely interpretation of this observation. Structural and kinetic similarities between pepsin and other aspartic proteinases, including the therapeutically important targets HIV protease and renin, suggest a similar slow reprotonation step after catalysis. This mechanistic feature has important implications regarding inhibitor design; if most of the enzymes are present in a product-release form during steady-state turnover, then perhaps inhibitors should be designed as product analogs instead of substrate analogs.     

Novel peptide-based pepsin inhibitors containing an epoxide group

1,2-Epoxy-3-(p-nitrophenoxy)propane (EPNP) is known to inhibit pepsin A and other aspartic proteinases by reacting with the active site aspartic acid residue(s). However, the reaction is considerably slow in general, and therefore, it is desirable to develop similar reagents that are capable of inhibiting these enzymes more rapidly. In the present study, we synthesized a series of novel inhibitors which have a reactive epoxide group linked with peptide by a hydrazide bond, with a general structure: Iva-L-Val-L-Val-(L-AA)(n)-N2H2-ES-OEt (n = 0 approximately 2) (Iva, isovaleryl; AA, bulky hydrophobic or aromatic amino acid residue; ES, epoxysuccinyl). These inhibitors were shown to inhibit porcine pepsin A remarkably faster than EPNP.     

Analysis of binding interactions of pepsin inhibitor-3 to mammalian and malarial aspartic proteases

The nematode Ascaris suum primarily infects pigs, but also causes disease in humans. As part of its survival mechanism in the intestinal tract of the host, the worm produces a number of protease inhibitors, including pepsin inhibitor-3 (PI3), a 17 kDa protein. Recombinant PI3 expressed in E. coli has previously been shown to be a competitive inhibitor of a subgroup of aspartic proteinases: pepsin, gastricsin and cathepsin E. The previously determined crystal structure of the complex of PI3 with porcine pepsin (p. pepsin) showed that there are two regions of contact between PI3 and the enzyme. The first three N-terminal residues (QFL) bind into the prime side of the active site cleft and a polyproline helix (139-143) in the C-terminal domain of PI3 packs against residues 289-295 that form a loop in p. pepsin. Mutational analysis of both inhibitor regions was conducted to assess their contributions to the binding affinity for p. pepsin, human pepsin (h. pepsin) and several malarial aspartic proteases, the plasmepsins. Overall, the polyproline mutations have a limited influence on the Ki values for all the enzymes tested, with the values for p. pepsin remaining in the low-nanomolar range. The largest effect was seen with a Q1L mutant, with a 200-fold decrease in Ki for plasmepsin 2 from Plasmodium falciparum (PfPM2). Thermodynamic measurements of the binding of PI3 to p. pepsin and PfPM2 showed that inhibition of the enzymes is an entropy-driven reaction. Further analysis of the Q1L mutant showed that the increase in binding affinity to PfPM2 was due to improvements in both entropy and enthalpy.     

Cleavage specificities of aspartic proteinases toward oxidized insulin B chain at different pH values

The cleavage specificities of typical aspartic proteinases: pepsin A, gastricsin, cathepsin D and rhizopuspepsin, were examined at different pH values with oxidized insulin B chain as a substrate with special attention to the specificities near neutral pH. Significant differences in relative specificity for scissile bonds were observed between pH 2.0 and 5.5-6.5, which may be partly related with the changes in dissociation states of the His and Glu residues in the substrate and the ionizable residues in the active site of each enzyme.     

A systematic series of synthetic chromophoric substrates for aspartic proteinases.

The hydrolysis of the chromogenic peptide Pro-Thr-Glu-Phe-Phe(4-NO2)-Arg-Leu at the Phe-Phe(4-NO2) bond by nine aspartic proteinases of animal origin and seven enzymes from micro-organisms is described [Phe(4-NO2) is p-nitro-L-phenylalanine]. A further series of six peptides was synthesized in which the residue in the P3 position was systematically varied from hydrophobic to hydrophilic. The Phe-Phe(4-NO2) bond was established as the only peptide bond cleaved, and kinetic constants were obtained for the hydrolysis of these peptide substrates by a representative selection of aspartic proteinases of animal and microbial origin. The value of these water-soluble substrates for structure-function investigations is discussed.     

The structure of pepsin. II. Structure of the enzyme active site (at 2 angstroms resolution)

Detailed structure of the pepsin active site in the region of the active aspartic acid residues and substrate binding S1 and S1' sites is considered. At the active site of the enzyme crystals studied several molecules of ethanol were detected, which interact with active groups. The catalytic properties of aspartyl proteinases towards dipeptide substrates were explained on the base of the specific structure of S1 and S1' binding sites.     

Various aspects of structural studies of aspartate proteinases

The paper is a brief account of aspartic proteinases' structural studies developed in V.A. Engelhardt Institute of Molecular Biology during the last 3 years. The work on porcine pepsin has been finalized after the refinement of the monoclinic crystal form at 1.8 A resolution performed in collaboration with the group of protein structure and function studies of the University of Alberta in Canada. An important structural property of chymosin which explains the enzyme specificity has been found. Protein engineering work on chymosin is being developed. The structural template for aspartic proteinases has been elucidated and on the basis of this template the model of HIV-1 protease molecule has been built. Some approaches to the design of HIV-1 protease inhibitors were elucidated.