Pepsin Molecule Research Articles

Kinetics and Mechanism of Pepsinogen Activation

The spontaneous and pepsin-catalyzed activation of pepsinogen has been observed and analyzed kinetically. At appropriate protein concentrations (1 mg per ml or less), a kinetically first order reaction was observed in the pH range 1 to 3, implying an intramolecular activation mechanism. Substantiation of the first order reaction came from a linear plot of log pepsinogen concentration versus time, with an ordinate intercept at the log of the initial pepsinogen concentration. Moreover a 10-fold dilution in protein concentration did not diminish the half-life of the activation reaction. The first order rate constant of 4.7 per min at 28° was comparable to the kcat values observed for peptic hydrolysis of synthetic peptide substrates. At pH 4, we observed kinetics consistent with a mixed reaction mechanism involving the bimolecular reaction of a pepsin molecule and a pepsinogen molecule as well as an intramolecular activation reaction. Below pH 3, this second order process was studied by observing the initial rate of pepsinogen activation in the presence of pepsin. When the pH profiles of the first and second order rate constants were compared, the first order rate constant declined much more rapidly as pH increased than did the second order rate constant. This fact explains the change from a predominantly intermolecular activation mechanism at pH 4 to an intramolecular activation below pH 3.

Specific and Irreversible Inactivation of Pepsin by Substrate-like Epoxides

Abstract Several substrate-like epoxides were found to act as specific and irreversible inactivators of pepsin. Of these, 1,2-epoxy-3-(p-nitrophenoxy) propane (EPNP) was the most potent. When EPNP reacted with pepsin, all enzyme activity was lost. Two molecules of EPNP were found to be covalently bound to each molecule of completely inactivated enzyme. In this reaction, two carboxyl groups of the enzyme are each apparently esterified by one hydroxyl of glycol formed from EPNP. The esterification of carboxylates by epoxides is a well established reaction. The modified enzyme, EPNP-pepsin, released the bound EPNP groups in alkaline solution. On thin layer chromatography, the released modifying was identified as the glycol of EPNP. These observations support the view that esterification was the inactivation reaction. Prior reaction of pepsin with diazoacetyl-dl-norleucine methyl ester or p-bromophenacyl bromide was still followed by the reaction of 2 EPNP residues per pepsin molecule. The EPNP-reactive carboxyl must, therefore, differ from those carboxyl groups reacted with diazoacetyl-dl-norleucine methyl ester and p-bromophenacyl bromide. EPNP failed to inactivate pepsinogen. Moreover, in the presence of synthetic substrates or their hydrolytic products, the inactivation of pepsin by EPNP was significantly slower. Results on the EPNP incorporation in the presence and absence of substrates indicated that only one of the two modified carboxyl groups was protected by the presence of the dipeptide substrate. This active center carboxyl group may participate in the catalysis of the enzyme. The second EPNP-reactive carboxyl was apparently not essential for enzyme activity. EPNP was found to inactivate other gastric proteases: human gastricsin, human pepsin, and bovine rennin. The stoichiometry of the reaction in human gastricsin and pepsin was identical with that in porcine pepsin.

Amino acid sequence of C-terminal fragment of hog pepsin.

The C‐terminal fragment of hog pepsin, which had been obtained in the preceding study on the cyanogen bromide hydrolysate of this protein, was subjected to sequential studies. The 37‐residue peptide was digested in independent experiments by trypsin, chymotrypsin, and pepsin, and the amino acid sequence of the arising peptides was determined. In parallel experiments the sequence of seventeen amino acid residues in the N‐terminal region of the peptide was established by the Edman degradation technique. The obtained data permit the C‐terminal peptide of hog pepsin to be ascribed the amino acid sequence Asp‐Val‐Pro‐Thr‐Ser‐Ser‐Gly‐Glu‐Leu‐Trp‐Ile‐Leu‐Gly‐Asp‐Val‐Phe‐Ile‐Arg‐Gln‐Tyr‐Tyr‐Thr‐Val‐Phe‐Asp‐Arg‐Ala‐Asn‐Asn‐Lys‐Val‐Gly‐Leu‐Ala‐Pro‐Val‐Ala. These results extend and partly correct our previous knowledge of this region of the pepsin molecule as recorded in literature. The problem of the tryptophan content of pepsin is briefly discussed.

A reactive aspartyl residue of pepsin

Recently it was reported ( Hamilton, Spona, and Crowell, 1967) that pepsin was inactivated by an equimolar amount of 1-diazo-4-phenylbutanone-2 (DPB). The results indicated that the reaction occurred at or near the active site of pepsin. In the present communication we report evidence indicating that DPB reacts with pepsin to form an ester of 1-hydroxy-4-phenylbutanone-2 (HPB) with the β-carboxyl group of an aspartyl residue, and that the amino acid sequence containing this aspartyl residue is: Ile-Val-Asp-Thr. This aspartyl residue is different from that attacked by another class of pepsin inhibitors, the α-haloketones ( Erlanger, Vratsanos, Wassermann, and Cooper, 1966). Therefore, the present results define a different section of the pepsin molecule which may be involved in the enzymic catalysis.

The Inactivation of Pepsin by Diazoacetylnorleucine Methyl Ester

Abstract Diazoacetyl-dl-norleucine methyl ester rapidly inactivates pepsin. In the presence of cupric ions, only 1 eq of norleucine is introduced per molecule of pepsin. In the absence of cupric ions, the reaction is much slower, inactivation is not complete, and more than 1 eq of norleucine is incorporated. Little or no reaction occurs with pepsinogen or with pepsin previously inactivated by exposure to pH 8. The norleucine introduced into pepsin can be removed by treatment with hydroxylamine. Thus, there is reason to conclude that a single carboxyl group at the active site of pepsin may have been esterified by this reagent.

The Structure of Surface-denatured Protein. VII. The Activity and the Shape of the Surface-denatured Pepsin Molecule

Abstract (1) The molecular weight, the size and the shape of the pepsin molecule on the substrates of pH 2 and 3 were determined. The molecule on the substrate of pH 3 (its isoelectric point) was of rather a round shape, but the molecule on pH 2 had a far more elongated shape. (2) The enzymatic activity of the surfacedenatured pepsin molecule on the solutions of various pHs was determined. The roundshaped molecule on the substrate of pH 3 was the most active, but the elongated molecule on pH 2 retained less activity.

On the surface inactivation of pepsin

1. 1. A new method is described for the isolation of unimolecular films of protein. 2. 2. This method has been applied to the study of films of pepsin. The results suggest that a fully unfolded pepsin film possesses no enzymic activity. There is no reason to suppose that a pepsin molecule, after having undergone complete unfolding at an air-water interface, can readily regain an active configuration.

Infra-Red Spectra of Films of Native and Denatured Pepsin

WE report here some observations on the infrared absorption of films prepared by drying native, denatured, and regenerated pepsin solutions. On thermodynamic grounds1 the alkali denaturation of pepsin molecules unrestricted in space (dilute aqueous solution) would be expected to involve the breaking of a large number of intra-molecular hydrogen bonds. As yet, direct evidence as to the formation of new intermolecular hydrogen bonds between uncoiled molecules is lacking. Differences in the infra-red absorption spectra of native and denatured pepsin in the range of hydrogen-bonding frequencies can, with reasonable assurance, be assumed to represent evidence for overall changes in hydrogen bonding. The infra-red spectra of films of pepsin and other enzymes measured in the wave-length region 2–15 microns2,3 show, in addition to the strong bands around 3, 6 and 6.5 microns always obtained with proteins and polypeptides, several less intense absorption bands in both the 3-micron region and at wave-lengths longer than 6.6 microns. In this communication we are concerned only with the region 3,400–2,800 cm.−1 (2.9–3.6 microns), in which the spectral measurements were made using calcium fluoride and lithium fluoride prisms, since this is a region of absorption by hydrogen-bonding frequencies.

ACETYLATION OF TYROSINE IN PEPSIN

Crystalline 60 per cent active acetyl pepsin has 7 acetyl groups per mol of pepsin, 3 of which are readily hydrolyzed in acid at pH 0.0 or in weak alkali at pH 10.0. The tyrosine-tryptophane content of this acetylated pepsin, measured colorimetrically, is less than pepsin by three tyrosine equivalents. Hydrolysis at pH 0.0 or pH 10.0 of the 3 acetyl groups results in a concomitant increase in the number of tyrosine equivalents. In the pH 0.0 hydrolysis experiment there is also a simultaneous increase in specific activity. The phenol group of glycyl tyrosine is acetylated by ketene under the conditions used in the acetylation of pepsin and the effect of pH on the rate of acetylation is similar in the two cases. It is concluded that the acetyl groups in the 60 per cent active acetyl pepsin, which are responsible for the decrease in specific enzymatic activity, are 3 in number and are attached to 3 tyrosine phenol groups of the pepsin molecule.