Monkey pepsinogens and pepsins. VI. One-step activation of Japanese monkey pepsinogen to pepsin

When Japanese monkey pepsinogen was activated at pH 2.0 in the absence of pepstatin, the activation segment of the amino(N)-terminal 47 residues was released as a single intact polypeptide. This clearly shows that the pepsinogen was activated to pepsin directly. This direct activation was called a 'one-step' process. On the other hand, when pepsinogen was activated at pH 2.0 in the presence of pepstatin, an appreciable amount of pepsinogen was converted to an intermediate form between pepsinogen and pepsin, although a part of pepsinogen was activated directly to pepsin. The intermediate form was generated by releasing the N-terminal 25 residues of pepsinogen. This activation through the intermediate form is thought to be a 'two-step' or 'stepwise-activating' process involving a bimolecular reaction between pepstatin-bound pepsinogen and free pepsin.     

Isolation and characterization of pepsinogen from Trimeresurus flavoviridis (Habu snake)

Pepsinogen was isolated from the gastric mucosa of Trimeresurus flavoviridis (Habu snake) by DEAE-cellulose and DEAE-Sepharose ion-exchange chromatographies, and Sephacryl S-200 gel-chromatography. The yield calculated from the crude extract was 29% with 6.2-fold purification. The purified pepsinogen gave a single band on both native- and SDS-PAGE. As no other active enzyme was detected on the chromatographies, it was concluded that the Habu snake has one major pepsinogen. The molecular mass of the pepsinogen was estimated to be 38 kDa by SDS-PAGE. The sequence of the N-terminal 26 amino acid residues was determined and compared with those of other pepsinogens. The N-terminal structure of Habu snake pepsinogen was more homologous with those of mammalian pepsinogens C than those of mammalian pepsinogens A. The pepsinogen was rapidly converted to pepsin by way of an intermediate form induced by acidification. The optimum pH of Habu snake pepsin for bovine hemoglobin was 1.5-2.0, and it retained full activity at pH 6.2 and 30 degrees C on incubation for 30 min. The optimum temperature for the snake pepsin was 50 degrees C and it was stable at 40 degrees C on incubation for 10 min. The proteolytic activity of the pepsin toward bovine hemoglobin was about two times higher than that of porcine pepsin A, however, the activity toward oxidized bovine insulin B-chain was lower than that of porcine pepsin A, and it did not hydrolyze oligopeptides. The specificity for oxidized bovine insulin B-chain of the pepsin was different from that of porcine pepsin A. Habu snake pepsin was inhibited by pepstatin A but not by serine, cysteine, or metallo protease inhibitors.     

Isolation of an activation intermediate and determination of the amino acid sequence of the activation segment of human pepsinogen A

Upon activation of human pepsinogen A at pH 2.0 in the presence of pepstatin, an intermediate form was generated together with pepsin A. This activation intermediate could be separated from pepsinogen A and pepsin A by DE-32 cellulose chromatography at pH 5.5. It had a molecular weight intermediate between those of pepsinogen A and pepsin A, and contained about half the number of basic amino acid residues in pepsinogen A. It had phenylalanine as the amino(N)-terminal amino acid, and was deduced to be generated by release of N-terminal 25 residue segment from pepsinogen A. Amino acid sequence determination of the N-terminal portions of pepsinogen A and the intermediate from enabled us to elucidate the entire acid sequence of the 47-residue activation peptide segment as follow: [Formula: see text]. On the other hand, upon activation of pepsinogen A at pH 2.0 in the absence of pepstatin, cleavage of the activation segment occurred at several additional bonds. In addition, upon activation both in the presence and in the absence of pepsitatin, an additional activation intermediate, designated pepsin A', was formed in minor quantities. This form was identical with pepsin A, except that it had an additional Pro-Thr-Leu sequence preceding the N-terminal valine of pepsin A.     

Evidence for the occurrence of one-step activation in porcine pepsinogen

Upon activation at pH 2.0 and 14°C, a significant portion of porcine pepsinogen was found to be converted directly to pepsin, releasing the 44-residue intact activation segment. The released segment was further cleaved to smaller peptides at pH 2.0, but at pH 5.5 it formed a tight complex with pepsin, and the complex was chromatographically indistinguishable from pepsinogen. This intact segment could be isolated for the first time. Thus one-step activation occurs in porcine pepsinogen along with the already known sequential activation.     

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.     

Purificaton and characterization of a pepsinogen and its pepsin from proventriculus of the Japanese quail

A crude extract of the proventriculus of the Japanese quail gave at least five bands of peptic activity at pH 2.2 on polyacrylamide gel electrophoresis. The main component, constituting about 40% of the total acid protease activity, was purified to homogeneity by hydroxyapatite and DEAE-Sepharose column chromatographies. At below pH 4.0, the pepsinogen was converted to a pepsin, which had the same electrophoretic mobility as one of the five bands of peptic activity present in the crude extract. The molecular weights of the pepsinogen and the pepsin were 40 000 and 36 000, respectively. Quail pepsin was stable in alkali up to pH 8.5. The optimal pH of the pepsin on hemoglobin was pH 3.0. The pepsin had about half the milk-clotting activity of purified porcine pepsin, but the pepsinogen itself had no activity. The hydrolytic activity of quail pepsin on N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine was about 1% of that of porcine pepsin. Among the various protease inhibitors tested, only pepstatin inhibited the proteolytic activity of the pepsin. The amino acid composition of quail pepsinogen was found to be rather similar to that of chick pepsinogen C, and these two pepsinogens possessed common antigenicity.     

Modification of swine pepsin and pepsinogen by p-nitrophenyldiazonium chloride

It was found that at pH 5.2 and 40-fold excess of p-nitrophenyldiazonium chloride the inhibitor incorporation into the porcine pepsin molecule involves 1.9 residues, one residue being bound to tyrosine 189. Besides, tyrosines 44, 113, 154 and 174 enter the reaction. Modified pepsin retains 25% of the native enzyme activity. In the pepsinogen molecule the degree of tyrosine 189 modification diminishes 5 times; of 1.5 inhibitor molecules incorporated into the protein 0.78 residues are bound to tyrosine 113. The potential proteolytic activity of modified pepsinogen towards haemoglobin cleavage makes up to 60% of the original one. It is concluded that the activation peptide in the pepsinogen molecule masks the substrate binding site bearing tyrosine 189, thus preventing its modification with p-nitrophenyldiazonium chloride. The activation peptide in the pepsinogen molecule is presumably located in the vicinity of the wide loop bend carrying tyrosine residue 113, which may be the reason for the decreased pKa value of this residue and of its increased reactivity in the azocoupling reaction.     

Cloning of the authentic bovine gene encoding pepsinogen a and its expression in microbial cells

Bovine pepsin is the second major proteolytic activity of rennet obtained from young calves and is the main protease when it is extracted from adult animals, and it is well recognized that the proteolytic specificity of this enzyme improves the sensory properties of cheese during maturation. Pepsin is synthesized as an inactive precursor, pepsinogen, which is autocatalytically activated at the pH of calf abomasum. A cDNA coding for bovine pepsin was assembled by fusing the cDNA fragments from two different bovine expressed sequence tag libraries to synthetic DNA sequences based on the previously described N-terminal sequence of pepsinogen. The sequence of this cDNA clearly differs from the previously described partial bovine pepsinogen sequences, which actually are rabbit pepsinogen sequences. By cloning this cDNA in different vectors we produced functional bovine pepsinogen in Escherichia coli and Saccharomyces cerevisiae. The recombinant pepsinogen is activated by low pH, and the resulting mature pepsin has milk-clotting activity. Moreover, the mature enzyme generates digestion profiles with alpha-, beta-, or kappa-casein indistinguishable from those obtained with a natural pepsin preparation. The potential applications of this recombinant enzyme include cheese making and bioactive peptide production. One remarkable advantage of the recombinant enzyme for food applications is that there is no risk of transmission of bovine spongiform encephalopathy.     

Conversion of pepsinogen to pepsin. Further evidence for intramolecular and pepsin-catalyzed activation.

Exposure of pepsinogen to acid for less than 2 min yields a product with proteolytic activity. This activity is due to intramolecular and intermolecular formation of pepsin from pepsinogen. We find no evidence for intermolecular proteolytic activity in the zymogen. These conclusions are based upon two sets of experiments. First, chemical cleavage of pepsinogen during short activation is demonstrated by quantitative analysis of the NH2-terminal 2 residues of the pepsin and pepsinogen in an activation mixture. In addition, quantitative NH2-terminal analyses after activation under different conditions confirm our previous inference that the product of unimolecular pepsinogen activation is homogeneous whereas bimolecular activation produces a pepsin product with a variety of NH2 termini. Second, spectral changes which occur upon acidification of a pepsinogen solution and are reversed by neutralization are shown to be consistent with the chemical cleavage of pepsinogen during acidification. The first order rate constant for pepsinogen activation, calculated from these spectral experiments, agrees well with the value we had determined previously.     

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.     

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.     

Studies on the irreversible step of pepsinogen activation

The bond cleavage step of pepsinogen activation has been investigated in a kinetic study in which the denatured products of short-term acidifications were separated on SDS-polyacrylamide gels and the peptide products were quantitated by densitometry. Although several peptide products were observed, under the conditions of the experiments (pH values between 2.0 and 2.8, 22 degrees C), the only one that was a product of an initial bond cleavage was the 44-residue peptide, which upon removal from pepsinogen yields pepsin. The rate constant for this bond cleavage is 0.015 s-1 at pH 2.4, which is the same as that at which the alkali-stable potential activity of pepsinogen had been found to convert to the alkali-labile activity of pepsin. When the conversion of zymogen to enzyme was followed by the change in fluorescence of adsorbed 6-(p-toluidinyl)naphthalene-2-sulfonate (TNS), the rate of change in TNS fluorescence was the same as the conversion to alkali lability. However, pepstatin blocked the bond cleavage of pepsinogen to pepsin, but it permitted the fluorescence change to proceed. In fact, it accelerated the apparent rate of change of TNS fluorescence by shifting the pKa of an essential conjugate acid from 1.7 to 2.6. The conversion to alkali lability, therefore, may be considered to be a composite of a relatively slow conformational change (at the measured rate), followed immediately by a relatively fast bond cleavage.     

Occurrence of two different pathways in the activation of porcine pepsinogen to pepsin

Activation of porcine pepsinogen at pH 2.0 was found to proceed simultaneously by two different pathways. One pathway is the direct conversion process of pepsinogen to pepsin, releasing the intact activation segment. The isolation of the released 44-residue segment was direct evidence of this one-step process. At pH 5.5 the segment bound tightly to pepsin to form a 1:1 pepsin-activation segment complex, which was chromatographically indistinguishable from pepsinogen. The other is a stepwise-activating or sequential pathway, in which pepsinogen is activated to pepsin through intermediate forms, releasing activation peptides stepwisely. These intermediate forms were isolated and characterized. The major intermediate form was shown to be generated by removal of the amino-terminal 16 residues from pepsinogen. The released peptide mixture was composed of two major peptides comprising residues 1-16 and 17-44, and hence the stepwise-activating process was deduced to be mainly a two-step process.     

Crystallization and preliminary crystal data of porcine pepsinogen

Single crystals of porcine pepsinogen, suitable for x-ray diffraction studies, have been grown with lithium sulfate as the precipitant. These pepsinogen crystals were dissolved, activated, and assayed for proteolytic activity. The specific enzymic activity of the dissolved crystalline protein was nearly twice that of the commerical pepsinogen from which the crystals were grown. Incubation at pH 8 before assay demonstrated that the crystals are free of pepsin. This crystal form of pepsinogen belongs to the monoclinic space group C2 with 4 molecules in the unit cell. The unit cell dimensions are a = 104.8 +/- 0.5 A, b = 43.1 +/- 0.1 A, c = 88.4 +/- 0.3 A, and beta = 91.3 degrees.     

The purification and properties of a single chicken pepsinogen fraction and the pepsin derived from it

1. Evidence is given for the presence of at least five pepsinogens in a crude extract of mixed chicken stomachs. One of these was purified and could be activated to yield a single pepsin. 2. The molecular weights of the pepsinogen and pepsin were 36000 and 34000 respectively. The pepsin associated at low pH values and low ionic strength. 3. The amino acid analyses of both proteins are given. The pepsin was devoid of phosphate but contained carbohydrate. 4. The N-terminal amino acids of pepsinogen and pepsin were serine and threonine respectively. Five amino acids were released by carboxypeptidase A and it was deduced that serine may be the C-terminal one. 5. Each protein contained one thiol group per molecule as determined by titration with p-chloromercuribenzoate. The rate of the reaction was very rapid with pepsin, but much slower with pepsinogen, although the same group appeared to react in both instances. The enzymic activity of pepsin was unaffected by the modification. 6. The isoionic point of the pepsin was close to pH4.0 and the enzyme was stable for long periods at pH values up to 7.0. 7. The enzyme hydrolysed bisphenyl sulphite almost as rapidly as did pig pepsin A.     

Kinetics of autocatalytic zymogen activation measured by a coupled reaction: pepsinogen autoactivation

A kinetic study was performed of a model for an autocatalytic zymogen activation process involving both intra- and intermolecular routes, to which a chromogenic reaction in which the active enzyme acts upon one of its substrates was coupled to continuously monitor the reaction. Kinetic equations describing the evolution of species involved in the system with time were obtained. These equations are valid for any zymogen autocatalytic activation process under the same initial conditions. Experimental design and kinetic data analysis procedures to evaluate the kinetic parameters, based on the derived kinetic equations, are suggested. In addition, a dimensionless distribution coefficient was defined, which shows mathematically whether the intra- or the intermolecular route prevails once the kinetic parameters involved in the system are known. The validity of the results obtained was checked using simulated curves for the species involved. As an example of application of the method, the system is experimentally illustrated by the continuous monitoring of pepsinogen transformation to pepsin.     

The high-resolution crystal structure of porcine pepsinogen.

The structure of porcine pepsinogen at pH 6.1 has been refined to an R-factor of 0.173 for data extending to 1.65 A. The final model contains 180 solvent molecules and lacks density for residues 157-161. The structure of this aspartic proteinase zymogen possesses many of the characteristics of pepsin, the mature enzyme. The secondary structure of the zymogen consists predominantly of beta-sheet, with an approximate 2-fold axis of symmetry. The activation peptide packs into the active site cleft, and the N-terminus (1P-9P) occupies the position of the mature N-terminus (1-9). Thus changes upon activation include excision of the activation peptide and proper relocation of the mature N-terminus. The activation peptide or residues of the displaced mature N-terminus make specific interactions with the substrate binding subsites. The active site of pepsinogen is intact; thus the lack of activity of pepsinogen is not due to a deformation of the active site. Nine ion pairs in pepsinogen may be important in the advent of activation and involve the activation peptide or regions of the mature N-terminus which are relocated in the mature enzyme. The activation peptide-pepsin junction, 44P-1, is characterized by high thermal parameters and weak density, indicating a flexible structure which would be accessible to cleavage. Pepsinogen is an appropriate model for the structures of other zymogens in the aspartic proteinase family.     

Modification of lysine residues of pepsinogen with dicarboxylic anhydrides

Porcine pepsinogen was modified chemically with citraconic, maleic and itaconic anhydrides, and the properties of the derivatives were studied as to the nature and extent of the structural changes in the protein. Modification of the zymogen with citraconic and maleic anhydrides was shown to be partially reversible; the citraconyl and maleyl groups were readily removed under mild acidic conditions. At pH 2.0, 77% and 50% of the potential pepsin activity could be recovered on activation of the citraconyl- and maleylpepsinogens. Itaconylpepsinogen regained only 25% of the potential pepsin activity.     

Diethyl pyrocarbonate inactivates swine pepsinogen by reaction at amino groups

One of the histidine residues in swine pepsinogen can be modified in a rapid reaction with diethyl pyrocarbonate (DEP). At the same time, the potential proteolytic activity of the zymogen is reduced to 40% of its initial value. Reaction at the histine residue is reversed by neutral hydroxylamine, but the loss in activity is not. Fractionation of DEP-pepsinogen activation mixtures gave fully active pepsin in reduced yield, not modified enzyme with reduced specific activity. This was taken to indicate that the reaction had produced a mixture of products. Irreversible incorporation of [¹⁴C]DEP indicates that carbethoxy groups are incorporated into the molecule at amino acids other than histidine. The positions of these carbethoxy groups were determined by tryptic digestion and hplc. Modification was found to have occurred, in nonstoichiometric amounts, at lysine residues 3 and 9 and at leucine 1. Control experiments showed that activation was not affected by reaction at leucine 1, indicating that inactivation is caused by reaction at one or more of the lysine residues.     

A pepsinogen from rainbow trout

1. A pepsinogen, Ia on the basis of its electrophoretic mobility, from rainbow trout stomach, has an optimum pH near 2 for activation. 2. The cognate pepsin is denatured at pH values above 7, in contrast to the zymogen, which is slightly more alkali-stable. It has an optimum pH of 3 for proteolysis of denatured hemoglobin. 3. The intrinsic reactivity of the zymogen and pepsin (rates of activation and of proteolysis, respectively) are quite high, but as they operate at the environmental temperature of the fish, are remarkably similar to rates of activation and proteolysis by mammalian pepsinogens and pepsins.