N-terminal sequence of swine pepsinogen and pepsin. The site of pepsinogen activation

The sequence of 119 amino acids of swine pepsinogen comprising the fragment released during the zymogen activation as well as the N-terminal part of pepsin is established. The activation of swine pepsinogen is shown to be accompanied by specific cleavage of Leu-Ile bond in the sequence:

$\text{Ala}\text{41}\text{Ala Ala Leu Ile}\text{Gly}\text{46}$

where Ile-45 represents the N-terminal residue of pepsin. This sequence is attacked in the course of pepsinogen activation by external enzymes — neutral proteinases and elastase.     

The amino acid composition of crystalline pepsin

The amino acid composition of twice recrystallized pepsin (Worthington Biochemical Corporation) has been determined chromatographically on columns of Amberlite IR 120 resin. The results of the analyses obtained on four different preparations indicate a close agreement in their amino acid composition. Pepsin is unique in that it has a great predominance of acidic amino acids over basic ones. Moreover, all the preparations contain a small and constant amount of hydroxyproline, corresponding to about 0.1 residue per molecule.     

Enzymic dephosphorylation of pepsin and pepsinogen

It has been shown by the work presented in this paper that it is possible to dephosphorylate enzymically pepsin and pepsinogen with a variety of phosphatases. With the aid of a phosphodiesterase and the prostate phosphatase it has been established that the phosphorus in the two proteins is present as a diester and connects two sites of the peptide chain in a cyclic configuration. Removal of the phosphorus does not affect the proteolytic activity against hemoglobin or the synthetic substrate acetyl-L-phenylalanyl diiodotryosine, nor the pepsinogen pepsin transformation. However, an increase of the autodigestion of pepsin is observed.     

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.     

Conversion of pepsinogen into pepsin is not a one-step process.

By incubation of pepsinogen with pepstatin at pH2.5, the first 'active' protein generated on activation is trapped in an inactive complex. The first activation peptide liberated has been identified as residues 1-16 from the pepsinogen sequence. This suggests a sequential mechanism rather than a one-step formation of pepsin.     

On the apparent inhibition of intramolecular activation of pepsinogen by pepsin substrates.

Marciniszyn et al. (Marciniszyn, J., Huang, J. S. Hartsuch, J. A., Tang, J. (1976) J. Biol. Chem. 251, 7095-7102) have recently suggested an intermediate in the intramolecular activation of pepsinogen. As evidence, they showed apparent competitive inhibition of activation by globin, indication a pepsinogen-globin complex. Previous work had shown pepsinogen activation to occur very rapidly in the presence of high concentrations of hemoglobin, a very similar pepsin substrate (McPhie, P. (1974) Biochem. Biophys. Res. Commun. 56, 789-792). This contradiction has been resolved by a re-evaluation of the techniques used in the two investigations. The experimental conditions of Marciniszyn et al. Were inadequately defined to ensure denaturation of pepsin, a prerequisite of their method. A small decrease in pH, caused by the presence of extraneous protein, prevents this denaturation and leads to consistent underestimates of the rate of zymogen activation.     

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.