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.     

Activation of human pepsinogens

Human pepsinogen A3 and A5 have been purified to chromatographic and electrophoretic homogeneity. At pH 2 pepsinogen A3 activates at a much faster rate than pepsinogen A5. Leu-23-Lys-24 is the first bond cleaved during activation of pepsinogen A3. This bond is also cleaved in pepsinogen A5, but together with the cleavage of Asp-25-Phe-26. Amino acid sequencing shows that pepsinogen A3 has Glu at position 43, whereas pepsinogen A5 has Lys.     

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.     

Activation mechanism of monkey and porcine pepsinogens A. One-step and stepwise activation pathways and their relation to intramolecular and intermolecular reactions

The activation of Sepharose-bound monkey pepsinogen A under acidic conditions proceeded by cleavage of the Leu47-Ile48 bond, indicating the occurrence of the intramolecular one-step activation, although the rate of cleavage was very slow. On the other hand the activation of monkey pepsinogen A in solution was highly dependent on pepsinogen concentration and the addition of exogenous pepsin A accelerated the rate of activation, indicating the predominance of intermolecular reaction. The cleavage site, however, was also restricted to the Leu47-Ile48 bond. Thus, apparently exclusive one-step activation occurred in monkey pepsinogen. The activation of porcine pepsinogen A in solution was also dependent on pepsinogen concentration and the addition of exogenous pepsin A accelerated the rate of activation. The major cleavage site by the exogenously added pepsin was the Leu44-Ile45 bond. Therefore the site most susceptible to the intermolecular attacks was the bond connecting the activation segment and the pepsin moiety in both monkey and porcine pepsinogens. In porcine pepsinogen, however, a part of the zymogen was activated through the intermediate form, and an intramolecular reaction was suggested to be involved in the generation of this form. These results showed that in both pepsinogens A the intramolecular reaction occurred, first yielding pepsin A or the intermediate form, which then acted intermolecularly on the remaining pepsinogen or the intermediate form to complete the activation in a short time. A molecular mechanism for the activation reaction was proposed to explain consistently the experimental results.     

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)     

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.     

The role of extra- and intracellular calcium in pepsinogen extrusion by isolated gastric glands]

It was found that carbacholine stimulated pepsinogen extrusion by isolated guinea pig stomach glands which were incubated in Ca(2+)-free medium, containing EGTA (0.25 mM). This effect could be imitated by caffeine (10 mM), a specific activator of Ca2+ release from intracellular pools. Extracellular Ca2+ in the concentrations over 0.125 mM increased pepsinogen extrusion which was stimulated by carbacholine. The interdependence between the level of pepsinogen extrusion and Ca2+ concentration in the medium had S-shaped character. La3+ ions (10(-4) mM) inhibited pepsinogen extrusion already in the first minutes after its activation by carbacholine. When testing other cations (Sr2+, Mg2+, Ba2+) it was found that only Sr2+ had some influence on pepsinogen extrusion. Thus, it can be concluded that both intra- and extracellular Ca2+ take part in the activation of pepsinogen extrusion. Obviously the role of extracellular Ca2+ consists in the support of reactivity of stomach glands to the action of stimulators of secretion.     

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.     

The concentration of pepsinogen C in human semen and the physiological activation of zymogen in the vagina

The relationship between male infertility and the pepsinogen C content in semen has been investigated. The activation of the seminal pepsinogen C in the vagina has been studied under physiological conditions. Samples of semen from 48 vasectomized males and from 46 males of infertile couples were analyzed for pepsinogen C by radioimmunoassay. No correlation was found between the level of pepsinogen C and seminal characteristics, including sperm concentration, motility, and morphologic features. The mean concentration of pepsinogen C was 42.2 micrograms/ml; the first, second, and third quartile were 18.4, 29.6, and 57.6 micrograms/ml, respectively. No significant difference in the level of pepsinogen C was observed between semen of normal quality, semen of reduced quality, and semen with aspermia. Activation of pepsinogen C occurred within 3 h when semen was incubated at pH below 5.0 at 37 degrees C. Intravaginal activation was investigated in six experiments in which semen from two males was instilled in three females. In four experiments with two couples, post-coital activation was investigated. Pepsin C activity in vaginal fluid was detected an average of 3 h (range 2-5 h) and 5 h (4-7 h) after instillation or ejaculation, respectively. Vaginal pH had then been below 4.5 for approximately 1 h. Pepsin C activity was present in the vagina for more than 24 h thereafter. It is most likely that seminal pepsin C is without influence on the fertilizing spermatozoon. However, pepsin C may exert a local effect in the vagina by degrading seminal proteins, thus preventing an immunogenic response in females.     

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.     

Activation process of pepsinogen.

The activation process of pepsinogen was analyzed by a combination of computer simulation and experiment. In order to investigate in detail the behavior of the basic schemes proposed in the previous study, further computer simulations were conducted. Some experiments were performed based on the information obtained. The changes in the UV difference spectrum in the early stage was measured by the stopped-flow technique and the conversion of pepsinogen to pepsin [EC 3.4.23.1] was followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, on the basis of the experimental results, the most reasonable scheme was selected and modified. As a result, a scheme for the activation process of pepsinogen was obtained (Scheme 8). On the basis of the above analyses, it was assumed that the first step and the third step are pH-dependent based on the change in the UV spectrum, that the second step is a nonlinear reaction containing a looped reaction with a dimeric intermediate (in this step, peptide fragments are released and pepsinogen is converted to a pepsin-like molecule), and that the third step is an equilibrium reaction involving proton binding.     

Separation of porcine pepsinogen A and progastricsin. Sequencing of the first 73 amino acid residues in progastricsin.

Porcine pepsinogen A (EC 3.4.23.1) and progastricsin (EC 3.4.23.3) have been separated by chromatography on DEAE-cellulose followed by chromatography on DEAE-Sepharose. Agar gel electrophoresis at pH 6.0 showed the presence of three components of pepsinogen A and two of progastricsin. During activation at pH 2 a segment of 43 amino acid residues (the prosegment peptide) is cleaved from the N-terminus of progastricsin. The sequence of this was determined; in addition, the first 30 residues of gastricsin were sequenced. The sequence of the first 73 amino acid residues of progastricsin shows an overall identity with progastricsins from man, monkey and rat of 67%. The overall identity with other zymogens for gastric proteinases is 27%. The highly conserved Lys36p (pig pepsinogen A numbering) is changed to Arg in porcine progastricsin.     

Analysis of the activation of pepsinogen in the presence of protein substrates and estimation of the intrinsic proteolytic activity of pepsinogen

Monkey pepsinogen A, monkey progastricsin, and porcine pepsinogen A were activated in the presence of two different protein substrates, namely, reduced and carboxymethylated lysozyme and hemoglobin. In each case, an extensive delay in activation was observed. The intermolecular activation reaction required for the generation of pepsin or gastricsin was strongly inhibited and this inhibition was essentially responsible for the delay. However, the intramolecular reaction required for the generation of the intermediate forms of the proenzymes was scarcely affected. The delay was longer at pH 3.0 than at pH 2.0. Irrespective of the delay in activation of pepsinogen, the digestion of substrates proceeded rapidly, evidence of the significant proteolytic activity of pepsinogen itself. Kinetic experiments demonstrated that pepsinogen changed from an enzymatically inactive species to an active species before the release of the activation segment. The proteolytic activity of the active pepsinogen was highest at pH 2.0, at 37 degrees C and the activity under these conditions was comparable to that of pepsin.     

Kinetic studies on the unfolding and refolding of pepsinogen in urea. The nature of the rate-limiting step

Kinetic studies on the unfolding of pepsinogen by urea showed that changes in absorbance and potential pepsin activity followed simple first order kinetics. Changes in these variables on refolding were more complex. A large part of the absorbance was recovered within the mixing time of these experiments, whereas the appearance of activity was a slow sigmoidal function of time. The results were interpreted to show that pepsinogen can rapidly regain a globular form, but that its activatable form is produced by a slow conformational change in the folded protein. The enthalpies of activation of this change are similar to those of the cis-trans isomerization of proline residues. If the latter reaction is involved in the folding of pepsinogen, it must occur after extensive folding has already occurred.     

Conformational change that accompanies pepsinogen activation observed in real time by fluorescence energy transfer

Pig pepsinogen has been reacted with N-carboxymethylisatoic anhydride to form N-carboxymethyl-anthraniloyl-(CMA-) pepsinogen, derivatized at Lysp18, Lysp23, Lysp27, Lysp30, and Lys320. Conformational change associated with activation was detected by following energy transfer from tryptophan residues of the pepsin moiety, excited at 295 nm, to CMA groups, monitored by emission above 415 nm. Efficiency of this energy transfer is a measure of conformational change. For this zymogen derivative the change in efficiency occurs with a first order rate constant of 0.041 s-1 at pH 2.4, 22 degrees, which equals the rate at which, following acidification, alkali-stable potential activity becomes alkali-labile. For the native zymogen the rate of this conversion had been shown to be identical to the rate of cleavage of the scissile bond of pepsinogen. Therefore, the correspondence in this derivative of the rates of conversion to alkali lability and change in energy transfer demonstrates that a conformational change accompanies the peptide bond cleavage of activation.     

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.     

The complete amino acid sequence of monkey pepsinogen A

The complete amino acid sequence of pepsinogen A from the Japanese monkey (Macaca fuscata) was determined. After converting the pepsinogen to pepsin by activation, the pepsin moiety was reduced and carboxymethylated, cleaved by cyanogen bromide, and the amino acid sequences of the major fragments determined. These fragments were aligned with the aid of overlapping peptides isolated from a chymotryptic digest of intact pepsin. Since the sequence of the activation segment had been determined previously (Kageyama, T., and Takahashi, K. (1980) J. Biochem. (Tokyo) 88, 9-16), the 373-residue sequence of monkey pepsinogen A was established, consisting of the pepsin moiety of 326 residues and the activation segment of 47 residues. Three disulfide bridges and 1 phosphoserine residue were found to be present in the pepsinogen molecule. The molecular weight was calculated to be 40,027 including the phosphate group. Monkey pepsinogen A showed high homology with human (94% identity) and porcine (86% identity) pepsinogens A.     

Studies on the Mechanism of Activation of Pepsinogen

The mechanism of activation of pepsinogen was studied. It was found that no peptide bond cleavage occurred in the molecule of denatured pepsinogen at pH 2. It was inferred from this that a specific secondary and tertiary structure is formed in the molecule of pepsinogen in acid and that it might be necessary for the hydrolysis of the peptide bond. From the circular dichroism studies on pepsinogen and pepsin, it was found that there is a conformational change in the molecule of pepsinogen at pH 4.3~4.5 and that this change is followed by a gradual formation of pepsin.     

Pepsinogen activation by microbial proteinases

Serine proteinase and metalloproteinase of Asp. oryzae, extracellular metalloproteinase of L. pneumophila and chymotrypsin-like proteinase of S. rutgersensis can hydrolyze pepsinogen by converting it into pepsin (pH 5.0, 37 degrees C). The localization of the site of hydrolysis depends on the nature of the enzyme: serine proteinase from Asp. oryzae induces the synthesis of a mixture of 60% pepsin, 25% leucyl-pepsin and 15% alanyl-leucyl-pepsin; metalloproteinase of Asp. oryzae converts pepsinogen only into leucyl-pepsin, while metalloproteinase of L. pneumophila yields a mixture of 33% pepsin, 53% leucyl-pepsin and 14% alanyl-leucyl-pepsin. Thus, the region of the activating pepsinogen peptide--Ala 42P-Ile 1 bond--seems to the most probable site for hydrolysis by exogenous proteinases. This site contains a Leu 44P-Ile 1 bond which is subjected to intermolecular hydrolysis during autocatalytic activation of pepsinogen. The experimental results emphasize the importance of the intermolecular pathway of pepsinogen activation.     

The N-terminal sequence of rat pepsinogen

The amino-acid sequence of 96 residues in the N-terminal region of rat pepsinogen I was determined and the first 46 residues were found to constitute the activation peptide segment. There was high degree of homology between the activation segments of rat pepsinogen and some pepsinogens A (pig, cow, Japanese monkey and human). However, the number of residues substituted between rat and the other pepsinogens were considerably larger than those among pepsinogens A. In the N-terminal 24 residues of active pepsin, homology (88%) between rat pepsin and human gastricsin was higher than that (50%) between rat pepsin and pepsin A from human or pig. This strongly suggests that rat pepsin should be classified as pepsin C.