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.     

Oligosaccharide units of lysosomal cathepsin D from porcine spleen. Amino acid sequence and carbohydrate structure of the glycopeptides

The amino acid sequences near the glycosylation sites and the oligosaccharide structures have been determined for the lysosomal protease cathepsin D from porcine spleen. Cathepsin D light and heavy chains were separately digested with proteases and the glycopeptides were purified. A single sequence was constructed from the amino acid sequence of the light chain glycopeptides which is: Tyr-Asn-Ser-Gly-Lys-Ser-Ser-Thr-Tyr-Val-Lys-Asn(CH2O)-Gly-Thr-Thr-Phe. A single glycopeptide sequence was also obtained for the heavy chain: Lys-Gly-Ser-Leu-Asp-Tyr-His-Asn(CH2O)-Val-Thr-Arg-Lys-Ala-Tyr. The light chain sequence is homologous with the sequence of porcine pepsin from residues 56 to 71. The heavy chain sequence is homologous with the pepsin sequence from residues 176 to 189. Thus, the 2 oligosaccharide-linked asparagines in cathepsin D correspond to residues 67 and 183 in pepsin and other homologous aspartyl proteases. These positions are located on the surface of the crystal structures of aspartyl proteases. Five oligosaccharides linked to Asn-67 were separated and their structures determined with proton NMR. Four major oligosaccharides are structural variants from the high mannose-type having 3, 5, 6, and 7 mannoses, respectively. A minor structure contained a third GlcNAc. Three oligosaccharide structures were found linked to Asn-183. Two major oligosaccharides are of the high mannose-type each with 5 mannose residues. One of the two contains a fucose linked to a GlcNAc. A third, very minor oligosaccharide contains galactose.     

Evolution in the structure and function of aspartic proteases

Aspartic proteases (EC3.4.23) are a group of proteolytic enzymes of the pepsin family that share the same catalytic apparatus and usually function in acid solutions. This latter aspect limits the function of aspartic proteases to some specific locations in different organisms; thus the occurrence of aspartic proteases is less abundant than other groups of proteases, such as serine proteases. The best known sources of aspartic proteases are stomach (for pepsin, gastricsin, and chymosin), lysosomes (for cathepsins D and E), kidney (for renin), yeast granules, and fungi (for secreted proteases such as rhizopuspepsin, penicillopepsin, and endothiapepsin). These aspartic proteases have been extensively studied for their structure and function relationships and have been the topics of several reviews or monographs (Tang: Acid Proteases, Structure, Function and Biology. New York: Plenum Press, 1977; Tang: J Mol Cell Biochem 26:93-109, 1979; Kostka: Aspartic Proteinases and Their Inhibitors. Berlin: Walter de Gruyter, 1985). All mammalian aspartic proteases are synthesized as zymogens and are subsequently activated to active proteases. Although a zymogen for a fungal aspartic protease has not been found, the cDNA structure of rhizopuspepsin suggests the presence of a "pro" enzyme (Wong et al: Fed Proc 44:2725, 1985). It is probable that other fungal aspartic proteases are also synthesized as zymogens. It is the aim of this article to summarize the major models of structure-function relationships of aspartic proteases and their zymogens with emphasis on more recent findings. Attempts will also be made to relate these models to other aspartic proteases.     

A study of aspartyl proteases using intramolecularly quenched fluorogenic peptide substrates

A series of fluorogenic tetra-, penta-, and hexapeptide substrates of the general structure Abz-X-Phe-Phe-Y-Ded or (-pNa in place of -Ded), where X=Ala, Ala-Ala, or Val-Ala and Y=−, Ala, or Ala-Ala, were proposed. Kinetic parameters of hydrolysis of these substrates by pepsin, cathepsin D, human gastricsin, pig pepsin, calf chymosin, and aspergillopepsin A were determined. The compounds synthesized proved to be effective substrates for aspartyl proteases of diverse origins.     

A study of aspartyl proteases using intramolecularly quenched fluorogenic peptide substrates

A series of fluorogenic tetra-, penta-, and hexapeptide substrates of the general structure Abz-X-Phe-Phe-Y-Ded (or -pNa in place of -Ded), where X = Ala, Ala-Ala, or Val-Ala and Y = -, Ala, or Ala-Ala, were proposed. Kinetic parameters of hydrolysis of these substrates by pepsin, cathepsin D, human gastricsin, pig pepsin, calf chymosin, and aspergillopepsin A were determined. The compounds synthesized proved to be effective substrates for aspartyl proteases of diverse origins.     

Ddi1, a eukaryotic protein with the retroviral protease fold

Retroviral aspartyl proteases are homodimeric, whereas eukaryotic aspartyl proteases tend to be large, monomeric enzymes with 2-fold internal symmetry. It has been proposed that contemporary monomeric aspartyl proteases evolved by gene duplication and fusion from a primordial homodimeric enzyme. Recent sequence analyses have suggested that such "fossil" dimeric aspartyl proteases are still encoded in the eukaryotic genome. We present evidence for retention of a dimeric aspartyl protease in eukaryotes. The X-ray crystal structure of a domain of the Saccharomyces cerevisiae protein Ddi1 shows that it is a dimer with a fold similar to that of the retroviral proteases. Furthermore, the double Asp-Thr-Gly-Ala amino acid sequence motif at the active site of HIV protease is found with identical geometry in the Ddi1 structure. However, the putative substrate binding groove is wider in Ddi1 than in the retroviral proteases, suggesting that Ddi1 accommodates bulkier substrates. Ddi1 belongs to a family of proteins known as the ubiquitin receptors, which have in common the ability to bind ubiquitinated substrates and the proteasome. Ubiquitin receptors contain an amino-terminal ubiquitin-like (UBL) domain and a carboxy-terminal ubiquitin-associated (UBA) domain, but Ddi1 is the only representative in which the UBL and UBA domains flank an aspartyl protease-like domain. The remarkable structural similarity between the central domain of Ddi1 and the retroviral proteases, in the global fold and in active-site detail, suggests that Ddi1 functions proteolytically during regulated protein turnover in the cell.     

aspS encoding an unusual aspartyl protease from Sclerotinia sclerotiorum is expressed during phytopathogenesis

The gene aspS encoding an aspartyl protease has been cloned from Sclerotinia sclerotiorum by screening a genomic library with a PCR-amplified fragment of the gene. The open reading frame of 1368 bp interrupted by one intron would encode a preproprotein of 435 amino acids. The catalytic aspartyl residues characteristic of aspartyl proteases are conserved; however, the active-site motif (DSG) in the N-terminal lobe is unusual in that Ser replaced Thr used in the active-site motif (DTG) of the C-terminal lobe and in all other fungal aspartyl proteases. RT-PCR revealed that aspS expression in axenic culture is not subjected to catabolite repression and demonstrated that aspS is expressed from the beginning of infection of sunflower cotyledons.     

Amino acid sequence of porcine spleen cathepsin D light chain

The complete amino acid sequence of the light chain of cathepsin D from porcine spleen has been determined. The light chain consists of a single polypeptide chain with 97 amino acid residues. The sequence is: (formula; see text) The molecular weight of the light chain was calculated from this sequence to be 10,548 (without carbohydrates). A single disulfide bond links two half-cystine residues between positions 46 and 53. A cysteine residue is located at position 27. The light chain sequence is extensively homologous to the NH2-terminal sequence of other aspartyl proteases. It shows a 59% identity with the sequence of mouse submaxillary gland renin and a 49% identity with that of porcine pepsin. A single glycosylation site is located at residue 70 of the cathepsin D light chain. This site corresponds to position 67 of pepsin by homology. The active site aspartyl residue, corresponding to Asp-32 of pepsin, is located at residue 33 in the cathepsin D light chain.     

The pH-dependence of the binding of competitive inhibitors to pepsin

1. The pH-dependence of the binding to pepsin of four dipeptide competitive inhibitors is reported. Values of K(i) obtained from equilibrium-dialysis experiments agree closely with those from kinetic measurements. 2. The binding of uncharged N-acyl-dipeptide amides to pepsin is essentially independent of pH from 0.2 to 5.8. Values of K(i) for the corresponding N-acyl-dipeptide acids rise rapidly above pH3.5, and depend on the ionization of a group of apparent pK(a) 3.6. 3. The data indicate that pepsin does not undergo any gross conformation change (at least none that affects binding) over the whole pH range of its catalytic activity. The pH-dependence of the dipeptide acid inhibitors indicates that the acid anions do not bind to pepsin, presumably because of electrostatic repulsion between the inhibitor anion and a negative centre at or near the active site of the enzyme. 4. The binding of all four stereoisomers of N-acetylphenylalanylphenylalanine, of the depside analogues of the l-l- and d-l-compounds and of N-acetylglycyl-l-phenylalanine and N-acetyl-l-phenylalanylglycine was studied at pH2.2. 5. These results throw further light on the binding specificity of pepsin and on the charge nature of the active site of this enzyme.     

Aspartyl proteases in Caenorhabditis elegans. Isolation, identification and characterization by a combined use of affinity chromatography, two-dimensional gel electrophoresis, microsequencing and databank analysis.

Crude homogenates of the nematode Caenorhabditis elegans exhibit maximal proteolytic activity under acidic pH conditions. About 90% of this activity is inhibited by the oligopeptide pepstatin, which specifically inhibits the activity of aspartyl proteases such as pepsin, cathepsins D and E or renin. We have purified enzymes responsible for this proteolytic activity by a single-step affinity chromatography on pepstatin-agarose. Analysis of the purified fraction by 1D SDS gel electrophoresis revealed six bands ranging from 35 to 52 kDa. After electrotransfer to poly(vinylidene difluoride) membranes, all bands were successfully subjected to N-terminal microsequencing. On 2D gels, the purified protein bands split into 19 spots which, after renewed microsequencing, were identified as isoelectric variants of the six proteins already described. The N-termini obtained for these proteins could be correlated to genomic DNA sequences determined in the course of the C. elegans genome sequencing project. All these sequences were predicted to code for expressed proteins as collected in the WORMPEP database. Five of the six coding sequences identified in this study were found to contain the typical active-site consensus sequence of aspartyl proteases and displayed an overall amino acid identity between 25 and 66% as compared to aspartyl proteases from other organisms. In addition to the five aspartyl proteases detected at the protein level, we have identified the coding sequences for seven other enzymes of this protease family by a similarity search in the genomic DNA of C. elegans which has recently been completely sequenced.     

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.     

Bovine pepsinogens and pepsins. The sequence around a reactive aspartyl residue

The specific inhibitor, N-diazoacetylnorleucine methyl ester reacts stoicheiometrically with bovine pepsin resulting in a simultaneous loss of all enzymic activity. A peptide containing a modified aspartyl group was isolated from bovine pepsin labelled with (14)C-labelled inhibitor. The aspartic acid residue is presumed to be part of the active centre and is in the same heptapeptide sequence as in porcine pepsin: Ile-Val-Asp-Thr-Gly-Thr-Ser.     

Digestive proteases in bodies and faeces of the two-spotted spider mite,Tetranychus urticae

Digestive proteases of the phytophagous mite Tetranychus urticae have been characterised by comparing their activity in body and faecal extracts. Aspartyl, cathepsin B- and L-like and legumain activities were detected in both mite bodies and faeces, with a specific activity of aspartyl and cathepsin L-like proteases about 5- and 2-fold higher, respectively, in mite faeces than in bodies. In general, all these activities were maintained independently of the host plant where the mites were reared (bean, tomato or maize). Remarkably, this is the first report in a phytophagous mite of legumain-like activity, which was characterised for its ability to hydrolyse the specific substrate Z-VAN-AMC, its activation by DTT and inhibition by IAA but not by E-64. Gel free nanoLC–nanoESI-QTOF MS/MS proteomic analysis of mite faeces resulted in the identification of four cathepsins L and one aspartyl protease (from a total of the 29 cathepsins L, 27 cathepsins B, 19 legumains and two aspartyl protease genes identified the genome of this species). Gene expression analysis reveals that four cathepsins L and the aspartyl protease identified in the mite faeces, but also two cathepsins B and two legumains that were not detected in the faeces, were expressed at high levels in the spider mite feeding stages (larvae, nymphs and adults) relative to embryos. Taken together, these results indicate a digestive role for cysteine and aspartyl proteases in T. urticae. The expression of the cathepsins B and L, legumains and aspartyl protease genes analysed in our study increased in female adults after feeding on Arabidopsis plants over-expressing the HvCPI-6 cystatin, that specifically targets cathepsins B and L, or the CMe trypsin inhibitor that targets serine proteases. This unspecific response suggests that in addition to compensation for inhibitor-targeted enzymes, the increase in the expression of digestive proteases in T. urticae may act as a first barrier against ingested plant defensive proteins.     

NMR study of the inhibition of pepsin by glyoxal inhibitors: mechanism of tetrahedral intermediate stabilization by the aspartyl proteases

Z-Ala-Ala-Phe-glyoxal (where Z is benzyloxycarbonyl) has been shown to be a competitive inhibitor of pepsin with a Ki = 89 +/- 24 nM at pH 2.0 and 25 degrees C. Both the ketone carbon (R13COCHO) and the aldehyde carbon (RCO13CHO) of the glyoxal group of Z-Ala-Ala-Phe-glyoxal have been 13C-enriched. Using 13C NMR, it has been shown that when the inhibitor is bound to pepsin, the glyoxal keto and aldehyde carbons give signals at 98.8 and 90.9 ppm, respectively. This demonstrates that pepsin binds and preferentially stabilizes the fully hydrated form of the glyoxal inhibitor Z-Ala-Ala-Phe-glyoxal. From 13C NMR pH studies with glyoxal inhibitor, we obtain no evidence for its hemiketal or hemiacetal hydroxyl groups ionizing to give oxyanions. We conclude that if an oxyanion is formed its pKa must be >8.0. Using 1H NMR, we observe four hydrogen bonds in free pepsin and in pepsin/Z-Ala-Ala-Phe-glyoxal complexes. In the pepsin/pepstatin complex an additional hydrogen bond is formed. We examine the effect of pH on hydrogen bond formation, but we do not find any evidence for low-barrier hydrogen bond formation in the inhibitor complexes. We conclude that the primary role of hydrogen bonding to catalytic tetrahedral intermediates in the aspartyl proteases is to correctly orientate the tetrahedral intermediate for catalysis.     

Gastric proteases of the Greenland cod Gadus ogac. I. Isolation and kinetic properties

The zymogens of three gastric proteases of the Greenland cod (Gadus ogac) were isolated by exclusion chromatography and chromatofocusing. The cod zymogens were activated more rapidly at lower temperatures than porcine pepsinogen and, after activation, were further purified by exclusion chromatography. The cod proteases had more alkaline pH optima and were active over a wider range of pH than porcine pepsin. The specific activity of porcine pepsin on protein substrates was greater than that of the individual cod proteases. However, the cod proteases had cumulative activity on protein substrates that was greater than the sum of their individual activities. Cod protease 1 was active on pepsin-specific substrates, and cod proteases 2 and 3 were active as gastricsin-specific substrates. All three cod proteases had greater milk-clotting activity and hydrolysed hemoglobin to a greater extent than porcine pepsin. The Vmax and Km,app of the cod proteases were dependent upon the substrate, and Vmax/Km,app values of the cod proteases were generally lower than porcine pepsin. It is suggested that the cod proteases together exhibit broad substrate specificity and maintain activity over a wide range of conditions to enhance protein digestion in the cod stomach.     

A Structural Biology Approach to Understand Human Lymphatic Filarial Infection

The presence of aspartic protease inhibitor in filarial parasite Brugia malayi (Bm-Aspin) makes it interesting to study because of the fact that the filarial parasite never encounters the host digestive system. Here, the aspartic protease inhibition kinetics of Bm-Aspin and its NMR structural characteristics have been investigated. The overall aim of this study is to explain the inhibition and binding properties of Bm-Aspin from its structural point of view. UV-spectroscopy and multi-dimensional NMR are the experiments that have been performed to understand the kinetic and structural properties of Bm-Aspin respectively. The human aspartic proteases that are considered for this study are pepsin, renin, cathepsin-E and cathepsin-D. The results of this analysis performed with the specific substrate [Phe-Ala-Ala-Phe (4-NO₂)-Phe-Val-Leu (4-pyridylmethyl) ester] against aspartic proteases suggest that Bm-Aspin inhibits the activities of all four human aspartic proteases. The kinetics studies indicate that Bm-Aspin follows a competitive mode of inhibition for pepsin and cathepsin-E, non-competitive for renin and mixed mode for cathepsin-D. The triple resonance NMR experiments on Bm-Aspin suggested the feasibility of carrying out NMR studies to obtain its solution structure. The NMR titration studies on the interactions of Bm-Aspin with the proteases indicate that it undergoes fast-exchange phenomena among themselves. In addition to this, the chemical shift perturbations for some of the residues of Bm-Aspin observed from ¹⁵N-HSQC spectra upon the addition of saturated amounts of aspartic proteases suggest the binding between Bm-Aspin and human aspartic proteases. They also provide information on the variations in the intensities and mode of binding between the proteases duly corroborating with the results from the protease inhibition assay method.     

Ambient pH Is a Major Determinant in the Expression of Cuticle-Degrading Enzymes and Hydrophobin by Metarhizium anisopliae

Secretion of proteolytic and chitinolytic enzymes is a hallmark of infection processes of Metarhizium anisopliae in response to host (insect) cuticular signals. The regulation of these enzymes (subtilisin-like proteases [Pr1a and Pr1b], trypsin-like proteases [Pr2], metalloproteases, aspartyl proteases, aminopeptidase, and chitinases) and a hydrophobin was investigated by Northern analysis and/or enzyme assay. The production of each enzyme showed a differential expression pattern in response to ambient pH; enzymes were synthesized only at pHs at which they function effectively, irrespective of whether the medium contained an inductive cuticle substrate. Three aspartyl proteases (pH optimum, 3), and chitinase (pH optimum, 5) showed maximal accumulation at acidic pHs. The highest level of aminopeptidase (pH optimum, 7) was detected at pH 7. The highest levels of five metalloproteases (pH optima, ca. 7) were detected over the pH range 6 to 8. Two trypsins and several subtilisin-like Pr1 isoforms with pH optima of ca. 8 were produced only under alkaline conditions. Northern analysis of RNA species corresponding to seven cDNA sequences encoding proteases and chitinase confirmed that the ambient pH played a major role in gene expression of secreted proteins. Hydrophobin was expressed almost equally at pHs 5 and 8 but was not expressed at pH 3. During fungal penetration, the pH of infected cuticle rises from about 6.3 to 7.7. Consistent with pH regulation of enzyme production, serine and metalloproteases were produced in situ during infection, but no production of aspartyl proteases was found. We propose that the alkalinity of infected cuticle represents a physiological signal that triggers the production of virulence factors.     

Lysine derivatives as potent HIV protease inhibitors. Discovery, synthesis and structure-activity relationship studies

A screening assay program on HIV-protease was carried out on more than fifty commercially available N-protected amino acids and has revealed that those with a long side chain such as lysine, ornithine and arginine exhibited significant inhibition of HIV protease enzyme. The presence of an Fmoc group was found to be essential to obtain micromolar inhibitors and the addition of an alkyl group at the Nalpha-position resulted in the discovery of the lead compound 11 displaying a 5 nM inhibition constant. Although this new inhibitor series is not categorized among those mimicking the substrate with a non-hydrolyzable transition-state isoster, it was found very specific to inhibit HIV protease enzyme in comparison to the mammalian aspartyl proteases pepsin, renin and cathepsin. Furthermore, these inhibitors did not show any cytotoxicity at a concentration below 75 microM.     

Linear non-competitive inhibition of solubilized human gamma-secretase by pepstatin A methylester,L685458, sulfonamides,and benzodiazepines

Cerebral deposition of amyloid beta-protein (A beta) is believed to play a key role in the pathogenesis of Alzheimer's disease. Because A beta is produced from the processing of amyloid beta-protein precursor (APP) by beta- and gamma-secretases, these enzymes are considered important therapeutic targets for identification of drugs to treat Alzheimer's disease. Unlike beta-secretase, which is a monomeric aspartyl protease, gamma-secretase activity resides as part of a membrane-bound, high molecular weight, macromolecular complex. Pepstatin and L685458 are among several structural classes of gamma-secretase inhibitors identified so far. These compounds possess a hydroxyethylene dipeptide isostere of aspartyl protease transition state analogs, suggesting gamma-secretase may be an aspartyl protease. However, the mechanism of inhibition of gamma-secretase by pepstatin and L685458 has not been elucidated. In this study, we report that pepstatin A methylester and L685458 unexpectedly displayed linear non-competitive inhibition of gamma-secretase. Sulfonamides and benzodiazepines, which do not resemble transition state analogs of aspartyl proteases, also displayed potent, non-competitive inhibition of gamma-secretase. Models to rationalize how transition state analogs inhibit their targets by non-competitive inhibition are discussed.     

Isolation of two aspartyl proteases from Trichoderma asperellum expressed during colonization of cucumber roots

Trichoderma asperellum and cucumber seedlings were used as a model to study the modulation of Trichoderma gene expression during plant root colonization. Seedlings were grown in an aseptic hydroponics medium and inoculated with Trichoderma spore suspension. Proteins differentially secreted into the medium were isolated. Three major proteins of fungal origin were identified: two arabinofuranosidases (Abf1 and Abf2) and an aspartyl protease. Differential mRNA display was conducted on Trichoderma mycelia interacting and non-interacting, with the plant roots. Among the differentially regulated clones another aspartyl protease was identified. Sequencing of the genes revealed that the first aspartyl protease is a close homologue of PapA from T. harzianum and the other, of AP1 from Botryotinia fuckeliana. RT-PCR analysis confirms that the proteases are induced in response to plant roots attachment and are expressed in planta. papA, but not papB, is also induced in plate confrontation assays with the plant pathogen Rhizoctonia solani. These data suggest that the identified proteases play a role in Trichoderma both as a mycoparasite and as a plant opportunistic symbiont.