Rearranging the domains of pepsinogen.

Most eukaryotic aspartic protease zymogens are synthesized as a single polypeptide chain that contains two distinct homologous lobes and a pro peptide, which is removed upon activation. In pepsinogen, the pro peptide precedes the N-terminal lobe (designated pep) and the C-terminal lobe (designated sin). Based on the three-dimensional structure of pepsinogen, we have designed a pepsinogen polypeptide with the internal rearrangement of domains from pro-pep-sin (native pepsinogen) to sin-pro-pep. The domain-rearranged zymogen also contains a 10-residue linker designed to connect sin and pro domains. Recombinant sin-pro-pep was synthesized in Escherichia coli, refolded from 8 M urea, and purified. Upon acidification, sin-pro-pep autoactivates to a two-chain enzyme. However, the emergence of activity is much slower than the conversion of the single-chain zymogen to a two-chain intermediate. In the activation of native pepsinogen and sin-pro-pep, the pro region is cleaved at two sites between residues 16P and 17P and 44P and 1 successively, and complete activation of sin-pro-pep requires an additional cleavage at a third site between residues 1P and 2P. In pepsinogen activation, the cleavage of the first site is rate limiting because the second site is cleaved more rapidly to generate activity. In the activation of sin-pro-pep, however, the second site is cleaved slower than the first, and cleavage of the third site is the rate limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of an activation intermediate of cathepsin E

Cathepsin E is an intracellular, non-lysosomal aspartic protease expressed in a variety of cells and tissues. The protease has proposed physiological roles in antigen presentation by the MHC class II system, in the biogenesis of the vasoconstrictor peptide endothelin, and in neurodegeneration associated with brain ischemia and aging. Cathepsin E is the only A1 aspartic protease that exists as a homodimer with a disulfide bridge linking the two monomers. Like many other aspartic proteases, it is synthesized as a zymogen which is catalytically inactive towards its natural substrates at neutral pH and which auto-activates in an acidic environment. Here we report the crystal structure of an activation intermediate of human cathepsin E at 2.35A resolution. The overall structure follows the general fold of aspartic proteases of the A1 family, and the intermediate shares many features with the intermediate 2 on the proposed activation pathway of aspartic proteases like pepsin C and cathepsin D. The pro-sequence is cleaved from the protease and remains stably associated with the mature enzyme by forming the outermost sixth strand of the interdomain beta-sheet. However, different from these other aspartic proteases the pro-sequence of cathepsin E remains intact after cleavage from the mature enzyme. In addition, the active site of cathepsin E in the crystal is occupied by N-terminal amino acid residues of the mature protease in the non-primed binding site and by an artificial N-terminal extension of the pro-sequence from a neighboring molecule in the primed site. The crystal structure of the cathepsin E/pro-sequence complex, therefore, provides further insight into the activation mechanism of aspartic proteases.     

Primary structure of a precursor to the aspartic proteinase from Rhizomucor miehei shows that the enzyme is synthesized as a zymogen

In order to characterize the zymogen of the milk-clotting enzyme from Rhizomucor miehei, we constructed a cDNA library on pBR327 in Escherichia coli. Aspartic proteinase-specific recombinants were isolated by colony hybridization to a specific oligonucleotide mixture, and the cDNA sequence corresponding to a precursor form of the enzyme was determined. The deduced amino acid sequence shows that this secreted fungal proteinase is synthesized as a precursor. The first 22 amino acid residues in this precursor constitute a typical signal peptide. The amino acid sequence of the following 47-amino-acid-long prosegment shows homology to the prosegments from both the extracellular and intracellular vertebrate aspartic proteinases, and to the prosegments from the yeast and Mucor pusillus aspartic proteinases as well. These observations suggest that all aspartic proteinases are synthesized with a prosegment and that this prosegment is essential for the correct folding of all the mature enzymes. The active Rhizomucor miehei enzyme consists of 361 amino acid residues with a total molecular weight of 38,701. Clusters of identities around the active site cleft support the assumption that these proteinases have a common folding of their peptide chains. The disulphide bridges were localized in the fungal enzyme, and 2 N-glycosylation sites were identified.     

Refined structure of porcine pepsinogen at 1.8 A resolution

The molecular structure of porcine pepsinogen at 1.8 A resolution has been determined by a combination of molecular replacement and multiple isomorphous phasing techniques. The resulting structure was refined by restrained-parameter least-squares methods. The final R factor [formula: see text] is 0.164 for 32,264 reflections with I greater than or equal to sigma (I) in the resolution range of 8.0 to 1.8 A. The model consists of 2785 protein atoms in 370 residues, a phosphoryl group on Ser68 and 238 ordered water molecules. The resulting molecular stereochemistry is consistent with a well-refined crystal structure with co-ordinate accuracy in the range of 0.10 to 0.15 A for the well-ordered regions of the molecule (B less than 15 A2). For the enzyme portion of the zymogen, the root-mean-square difference in C alpha atom co-ordinates with the refined porcine pepsin structure is 0.90 A (284 common atoms) and with the C alpha atoms of penicillopepsin it is 1.63 A (275 common atoms). The additional 44 N-terminal amino acids of the prosegment (Leu1p to Leu44p, using the letter p after the residue number to distinguish the residues of the prosegment) adopt a relatively compact structure consisting of a long beta-strand followed by two approximately orthogonal alpha-helices and a short 3(10)-helix. Intimate contacts, both electrostatic and hydrophobic interactions, are made with residues in the pepsin active site. The N-terminal beta-strand, Leu1p to Leu6p, forms part of the six-stranded beta-sheet common to the aspartic proteinases. In the zymogen the first 13 residues of pepsin, Ile1 to Glu13, adopt a completely different conformation from that of the mature enzyme. The C alpha atom of Ile1 must move approximately 44 A in going from its position in the inactive zymogen to its observed position in active pepsin. Electrostatic interactions of Lys36pN and hydrogen-bonding interactions of Tyr37pOH, and Tyr90H with the two catalytic aspartate groups, Asp32 and Asp215, prevent substrate access to the active site of the zymogen. We have made a detailed comparison of the mammalian pepsinogen fold with the fungal aspartic proteinase fold of penicillopepsin, used for the molecular replacement solution. A structurally derived alignment of the two sequences is presented.     

Secretion by yeast of the zymogen form of Mucor rennin, an aspartic proteinase of Mucor pusillus, and its conversion to the mature form

The Mucor rennin gene encoding a prepro-form of the fungal aspartic proteinase from Mucor pusillus was expressed under the control of the yeast GAL7 promoter in Saccharomyces cerevisiae. An inactive zymogen of the enzyme with the 44-amino-acid pro-sequence was identified in the medium during the initial stage of cultivation. Processing of the purified zymogen to the mature enzyme proceeded autocatalytically under the acidic conditions. The rate of processing was accelerated by an increase in the concentration of the zymogen or addition of the mature enzyme. The in vitro processing was inhibited by inhibitors for the aspartic proteinases. The zymogen with no proteinase activity due to a mutation at the active site residue, Asp, was still processed at a relatively slower rate in a wild-type strain of yeast, but no processing occurred in the pep4-3 mutant strain of S. cerevisiae deficient in yeast proteinase A. Thus, Mucor rennin is excreted in a form of zymogen, which is then processed in the yeast secretion pathway mainly by the autocatalytic proteolysis but, alternatively, by a proteinase of yeast.     

Crystal structures of Aspergillus oryzae aspartic proteinase and its complex with an inhibitor pepstatin at 1.9A resolution

The X-ray structures of Aspergillus oryzae aspartic proteinase (AOAP) and its complex with inhibitor pepstatin have been determined at 1.9A resolution. AOAP was crystallized in an orthorhombic system with the space group P2(1)2(1)2(1) and cell dimensions of a=49.4A, b=79.4A, and c=93.6A. By the soaking of pepstatin, crystals are transformed into a monoclinic system with the space group C2 and cell dimensions of a=106.8A, b=38.6A, c=78.7A, and beta=120.3 degrees. The structures of AOAP and AOAP/pepstatin complex were refined to an R-factor of 0.177 (R(free)=0.213) and of 0.185 (0.221), respectively. AOAP has a crescent-shaped structure with two lobes (N-lobe and C-lobe) and the deep active site cleft is constructed between them. At the center of the active site cleft, two Asp residues (Asp33 and Asp214) form the active dyad with a hydrogen bonding solvent molecule between them. Pepstatin binds to the active site cleft via hydrogen bonds and hydrophobic interactions with the enzyme. The structures of AOAP and AOAP/pepstatin complex including interactions between the enzyme and pepstatin are very similar to those of other structure-solved aspartic proteinases and their complexes with pepstatin. Generally, aspartic proteinases cleave a peptide bond between hydrophobic amino acid residues, but AOAP can also recognize the Lys/Arg residue as well as hydrophobic amino acid residues, leading to the activation of trypsinogen and chymotrypsinogen. The X-ray structure of AOAP/pepstatin complex and preliminary modeling show two possible sites of recognition for the positively charged groups of Lys/Arg residues around the active site of AOAP.     

Cathepsin D propeptide: mechanism and regulation of its interaction with the catalytic core

Propeptide blocks the active site in the inactive zymogen of cathepsin D and is cleaved off during zymogen activation. We have designed a set of peptidic fragments derived from the propeptide structure and evaluated their inhibitory potency against mature cathepsin D using a kinetic assay. Our mapping of the cathepsin D propeptide indicated two domains in the propeptide involved in the inhibitory interaction with the enzyme core: the active site "anchor" domain and the N-terminus of the propeptide. The latter plays a dominant role in propeptide inhibition (nanomolar Ki), and its high-affinity binding was corroborated by fluorescence polarization measurements. In addition to the inhibitory domains of propeptide, a fragment derived from the N-terminus of mature cathepsin D displayed inhibition. This finding supports its proposed regulatory function. The interaction mechanisms of the identified inhibitory domains were characterized by determining their modes of inhibition as well as by spatial modeling of the propeptide in the zymogen molecule. The inhibitory interaction of the N-terminal propeptide domain was abolished in the presence of sulfated polysaccharides, which interact with basic propeptide residues. The inhibitory potency of the active site anchor domain was affected by the Ala38pVal substitution, a propeptide polymorphism reported to be associated with the pathology of Alzheimer's disease. We infer that propeptide is a sensitive tethered ligand that allows for complex modulation of cathepsin D zymogen activation.     

Prodomain processing of Asp1 (BACE2) is autocatalytic

Generation of the amyloid peptide through proteolytic processing of the amyloid precursor protein by beta- and gamma-secretases is central to the etiology of Alzheimer's disease. The highly elusive beta-secretase was recently identified as a transmembrane aspartic proteinase, Asp2 (BACE). The Asp2 homolog Asp1 (BACE2/DRAP) has also been reported to exhibit beta-secretase cleavage of amyloid precursor protein. Most aspartic proteinases are generated as inactive proenzymes, requiring removal of the prodomain to generate active proteinase. Here we show that prodomain processing of Asp1 occurs between Leu(62) and Ala(63) and is autocatalytic. Asp1 cleaved a maltose-binding protein-Asp1 prodomain fusion protein and a synthetic peptide at this site. Mutation of one of the conserved catalytic aspartic acid residues in the active site of Asp1 to asparagine (D110N) abolished this cleavage. Mutation of P(1)' and P(2)' residues in the substrate to phenylalanine reduced cleavage at this site. Asp1 expressed in cells was the mature form, and prodomain processing occurred intramolecularly within the endoplasmic reticulum/early Golgi. Interestingly, a proportion of mature Asp1 was expressed on the cell surface. When full-length Asp1(D110N) was expressed in COS-7 cells, it was not processed, suggesting that no other proteinase can activate Asp1 in these cells.     

Regulation of progastricsin mRNA levels in sea bass (Dicentrarchus labrax) in response to fluctuations in food availability

In this study the sea bass (Dicentrarchus labrax) pepsinogen C gene was isolated. The nucleotide sequences of all exons are presented. The organization of the gene is compatible with that of other aspartic proteinases. The predicted 388-residue amino acid (aa) sequence of sea bass pepsinogen C consists of a signal sequence of 16 amino acid residues, an activation peptide of 43 residues, and the mature pepsin of 329 residues containing the two characteristic active-site aspartic acids. We also analyzed fasting-induced changes in the expression of progastricsin mRNA, using real-time RT-PCR absolute quantification. Progastricsin mRNA copy number was downregulated under conditions of negative energy balance, such as starvation, and upregulated during positive energy balance, such as refeeding. These findings offer new information about the sea bass progastricsin gene and support a role of this gastric digestive enzyme in the regulation of food intake in sea bass.     

The precursor of secreted aspartic proteinase Sapp1p from Candida parapsilosis can be activated both autocatalytically and by a membrane-bound processing proteinase

Opportunistic pathogens of the genus Candida produce secreted aspartic proteinases (Saps) that play an important role in virulence. Saps are synthesized as zymogens, but cell-free culture supernatants of Candida spp. contain only mature Saps. To study the zymogen conversion, the gene encoding a precursor of C. parapsilosis proteinase Sapp1p was cloned, expressed in E. coli and the product was purified. When placed in acidic conditions, the precursor was autocatalytically processed, yielding an active proteinase. The self-activation proceeded through an intermediate product and the resulting enzyme was one amino acid shorter than the authentic enzyme. This truncation did not cause changes in proteinase activity or secondary structure compared to the authentic Sapp1p. Accurate cleavage of the pro-mature junction, however, required a processing proteinase. A crude membrane fraction prepared from C. parapsilosis cells contained an enzyme with Kex2-like activity, which processed the Sapp1p precursor at the expected site. The pro-segment appeared to be indispensable for Sapp1p to attain an appropriate structure. When expressed without the pro-segment, the Sapp1p mature domain was not active and had a lower content of alpha-helical conformation, as measured by circular dichroism. A similar effect was observed when a His(6)-tag was linked to the C-terminus of Sapp1p or its precursor.     

Crystal structure of plant aspartic proteinase prophytepsin: inactivation and vacuolar targeting.

We determined at 2.3 A resolution the crystal structure of prophytepsin, a zymogen of a barley vacuolar aspartic proteinase. In addition to the classical pepsin-like bilobal main body of phytepsin, we also traced most of the propeptide, as well as an independent plant-specific domain, never before described in structural terms. The structure revealed that, in addition to the propeptide, 13 N-terminal residues of the mature phytepsin are essential for inactivation of the enzyme. Comparison of the plant-specific domain with NK-lysin indicates that these two saposin-like structures are closely related, suggesting that all saposins and saposin-like domains share a common topology. Structural analysis of prophytepsin led to the identification of a putative membrane receptor-binding site involved in Golgi-mediated transport to vacuoles.     

Synthesis, purification, and active site mutagenesis of recombinant porcine pepsinogen

In order to carry out studies on structure and function relationships of porcine pepsinogen using site-directed mutagenesis approaches, the cDNA of this zymogen was cloned, sequenced, expressed in Escherichia coli, and the protein refolded, and purified to homogeneity. Porcine pepsinogen cDNA, obtained from a lambda gt10 cDNA library of porcine stomach contains 1364 base pairs. It contains leader, pro, and pepsin regions of 14, 44, and 326 residues, respectively. In addition, it also contains 5'- and 3'-untranslated regions. Four differences are present between the sequence deduced from the cDNA and the pepsinogen sequence determined previously by protein chemistry methods. Residues P19 (in the pro region) and 263 are asparagines in the cDNA sequence instead of aspartic acids. Isoleucine 230 is not present in the cDNA sequence and residue 242 is a tyrosine in the cDNA instead of an aspartic acid. Porcine pepsinogen cDNA was placed under the control of a tac promoter in a plasmid and expressed in E. coli. The synthesis of pepsinogen was optimized to about 50 mg/liter of culture. The recombinant (r-) pepsinogen, which was insoluble, was recovered by centrifugation, washed, dissolved in 6 M urea in Tris-HCl, pH 8, and refolded by rapid dilution. r-pepsinogen was purified to homogeneity after chromatography on Sephacryl S-300 and fast protein liquid chromatography on a monoQ column. r-pepsinogen contains an additional methionine residue at the NH2 terminus as compared to native (n-) pepsinogen. However, r- and n-pepsinogens are indistinguishable in their intramolecular activation constants. After activation, r- and n-pepsins have the same NH2-terminal sequences as well as Km values. Based on these data, r-pepsinogen was judged suitable for mutagenesis studies. A mutant pepsinogen (D32A) with the active site aspartic acid changed to an alanine was produced and purified. D32A-pepsinogen did not convert to pepsin in acid solution but it bound to pepstatin with an apparent KD of about 5 x 10(-10) M. D32A-pepsinogen possesses no detectable proteolytic activity. These results indicate that (i) intramolecular pepsinogen activation is accomplished by the pepsin active site, and (ii) unlike subtilisin (Carter, P., and Wells, J. A. (1988) Nature 332, 564-568), the active site mutant of pepsin is not enzymically active.     

Primary structure of zymogen of streptococcal proteinase

The complete amino acid sequence has been derived for the zymogen of streptococcal proteinase. The protein yielded a unique sequence containing 337 amino acids in a single polypeptide chain. The NH₂-terminal residue of the zymogen is aspartic acid and the COOH terminus is proline. The signal peptide commonly associated with the intracellular form of many proteins secreted from eukaryotic cells was absent from the zymogen sequence. The transformation of the zymogen to the enzyme under controlled conditions of proteolysis by trypsin and by streptococcal protease itself involves the removal of 84 amino acid residues from the NH₂ terminus of the zymogen. The zymogen-to-enzyme conversion is accompanied by a change in serological specificity. An intermediate, modified zymogen formed in the transformation process contains only 12 amino acid residues less than the zymogen but shows the serological reactivity of both the zymogen and the enzyme.     

Mechanism of intramolecular activation of pepsinogen. Evidence for an intermediate delta and the involvement of the active site of pepsin in the intramolecular activation of pepsinogen.

Intramolecular pepsinogen activation is inhibited either by pepstatin, a potent pepsin inhibitor, or by purified globin from hemoglobin, a good pepsin substrate. Also, pepsinogen at pH 2 can be bound to a pepstatin-Sepharose column and recovered as native zymogen upon elution in pH 8 buffer. Kinetic studies of the globin inhibition of pepsinogen activation show that globin binds to a pepsinogen intermediate. This interaction gives rise to competitive inhibition of intramolecular pepsinogen activation. The evidence presented in this paper suggests that pepsinogen is converted rapidly upon acidification to the pepsinogen intermediate delta. In the absence of an inhibitor, the intermediate undergoes conformational change to bind the activation peptide portion of this same pepsinogen molecule in the active center to form an intramolecular enzyme-substrate complex (intermediate theta). This is followed by the intramolecular hydrolysis of the peptide bond between residues 44 and 45 of the pepsinogen molecule and the dissociation of the activation peptide from the pepsin. Intermediate delta apparently does not activate another pepsinogen molecule via an intermolecular process. Neither does intermediate delta hydrolyze globin substrate.     

Adaptation of the behaviour of an aspartic proteinase inhibitor by relocation of a lysine residue by one helical turn

In addition to self-inhibition of aspartic proteinase zymogens by their intrinsic proparts, the activity of certain members of this enzyme family can be modulated through active-site occupation by extrinsic polypeptides such as the small IA3 protein from Saccharomyces cerevisiae. The unprecedented mechanism by which IA3 helicates to inhibit its sole target aspartic proteinase locates an i, i+4 pair of charged residues (Lys18+Asp22) on an otherwise-hydrophobic face of the amphipathic helix. The nature of these residues is not crucial for effective inhibition, but re-location of the lysine residue by one turn (+4 residues) in the helical IA3 positions its side chain in the mutant IA3-proteinase complex in an orientation essentially identical to that of the key lysine residue in zymogen proparts. The binding of the extrinsic mutant IA3 shows pH dependence reminiscent of that required for the release of intrinsic zymogen proparts so that activation can occur.     

Pepsinogen-like activation intermediate of plasmepsin II revealed by molecular dynamics analysis

Plasmepsins are pharmaceutically relevant aspartic proteases involved in haemoglobin degradation by the malaria causing parasites Plasmodium spp. They are translated as inactive proenzymes, with an elongated prosegment. On prosegment cleavage, plasmepsins undergo a series of hitherto unresolved conformational changes before becoming active. Here, the flexibility of plasmepsin and proplasmepsin and the activation process are investigated by multiple explicit water molecular dynamics simulations. The large N-terminal displacement and the interdomain shift from the proenzyme structure to active plasmepsin are promoted by essential dynamics sampling. An intermediate, stabilized by electrostatic interactions between the catalytic dyad and the N-terminus of mature plasmepsin, is observed along all activation trajectories. Notably, the stabilizing interactions in the activation intermediate of plasmepsin are similar to those in the X-ray structure of pepsinogen. In particular, the catalytic aspartates act as hydrogen bond acceptors for the N-terminal amino group and the Ser2 hydroxyl in plasmepsin, and the side chains of Lys36pro and Tyr9 in pepsinogen. The simulation results are used to suggest in vitro experiments to test the conformational transitions involved in the maturation of plasmepsin, and design small-molecule inhibitors.     

Penicillopepsin-JT2, a recombinant enzyme from Penicillium janthinellum and the contribution of a hydrogen bond in subsite S3 to k(cat)

The nucleotide sequence of the gene (pepA) of a zymogen of an aspartic proteinase from Penicillium janthinellum with a 71% identity in the deduced amino acid sequence to penicillopepsin (which we propose to call penicillopepsin-JT1) has been determined. The gene consists of 60 codons for a putative leader sequence of 20 amino acid residues, a sequence of about 150 nucleotides that probably codes for an activation peptide and a sequence with two introns that codes for the active aspartic proteinase. This gene, inserted into the expression vector pGPT-pyrG1, was expressed in an aspartic proteinase-free strain of Aspergillus niger var. awamori in high yield as a glycosylated form of the active enzyme that we call penicillopepsin-JT2. After removal of the carbohydrate component with endoglycosidase H, its relative molecular mass is between 33,700 and 34,000. Its kinetic properties, especially the rate-enhancing effects of the presence of alanine residues in positions P3 and P2' of substrates, are similar to those of penicillopepsin-JT1, endothiapepsin, rhizopuspepsin, and pig pepsin. Earlier findings suggested that this rate-enhancing effect was due to a hydrogen bond between the -NH- of P3 and the hydrogen bond accepting oxygen of the side chain of the fourth amino acid residue C-terminal to Asp215. Thr219 of penicillopepsin-JT2 was mutated to Ser, Val, Gly, and Ala. Thr219Ser showed an increase in k(cat) when a P3 residue was present in the substrate, which was similar to that of the wild-type, whereas the mutants Thr219Val, Thr219Gly, and Thr219Ala showed no significant increase when a P3 residue was added. The results show that the putative hydrogen bond alone is responsible for the increase. We propose that by locking the -NH- of P3 to the enzyme, the scissile peptide bond between P1 and P1' becomes distorted toward a tetrahedral conformation and becomes more susceptible to nucleophilic attack by the catalytic apparatus without the need of a conformational change in the enzyme.     

Primary structure of human proacrosin deduced from its cDNA sequence

cDNA clones encoding proacrosin, the zymogen of acrosin, were isolated from a human testis cDNA library by using a fragment of boar acrosin cDNA as a probe. Nucleotide sequencing of the longest cDNA clone has predicted that human proacrosin is synthesized with a 19-amino-acid signal peptide at the N-terminus. The cleavable signal sequence is followed by a 23-residue segment corresponding to the light chain and then by a 379-residue stretch that constitutes the heavy chain containing the catalytic site of the mature protease. The C-terminal portion of the deduced sequence for the heavy chain is very rich in proline residues, most of which are encoded by a unique repeat of CCCCCA. The active-site residues including histidine, aspartic acid, and serine are also predicted to be located at residues 69, 123, and 221, respectively.     

Proteinases participating in the processing and activation of prolegumain in primary cultured rat macrophages

The mammalian legumain is a recently identified lysosomal cysteine proteinase belonging to the clan CD and homologous to plant legumain. This enzyme has the characteristic of specifically hydrolyzing peptide bonds after asparagine residues. As in the case of papain-type cysteine proteinases, legumain is synthesized as an inactive zymogen, and processed into a mature form localized in lysosomes. However, the mechanism of its activation remains unclear. In this study, we analyze which types of proteinases may participate in the processing of legumain in rat primary cultured macrophages using various proteinase inhibitors after 24 h treatment with Bafilomycin A1, a vacuolar ATPase inhibitor. The processing of legumain in macrophages was accomplished by papain-type cysteine proteinases other than cathepsin B.     

Molecular characteristics of pepsinogen and pepsin from duck glandular stomach

1. Two procedures were developed for the preparation of duck pepsinogen, an enzyme from the family of aspartic proteases (EC 3.4.23.1) and its zymogen. 2. The amino acid composition, sugar content and the partial N- and C-terminal sequences of both the enzyme and the zymogen were determined. These sequences are highly homologous with the terminal sequences of chicken pepsin(ogen). 3. Duck pepsinogen and pepsin are unlike other pepsin(ogen)s in being relatively stable in alkaline media: pepsinogen is inactivated at pH 12.1, pepsin at pH 9.6. 4. Duck pepsin is inhibited by diazoacetyl-D,L-norleucine methyl ester (DAN), 1,2-epoxy-3(p-nitrophe-noxy)propane (EPNP), pepstatin and a synthetic pepsin inhibitor Val-D-Leu-Pro-Phe-Phe-Val-D- Leu. The pH-optimum of duck pepsin determined in the presence of synthetic substrate is pH 4. 5. Duck pepsin has a marked milk-clotting activity whereas its proteolytic activity is lower than that of chicken pepsin. 6. The activation of duck pepsinogen is paralleled by two conformational changes. The activation half-life determined in the presence of a synthetic substrate at pH 2 and 14 degrees C is 20 sec.