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The total sequence of 365 amino acid residues in bovine prochymosin is presented. Alignment with the amino acid sequence of porcine pepsinogen shows that 204 amino acid residues are common to the two zymogens. Further comparison and alignment with the amino acid sequence of penicillopepsin shows that 66 residues are located at identical positions in all three proteases. The three enzymes belong to a large group of proteases with two aspartate residues in the active center. This group forms a family derived from one common ancestor.Chymosin (EC 3.4.23.4) is the major proteolytic enzyme in the stomach of the preruminant calf. Like pepsin, chymosin belongs to a group of acidic proteases in which two aspartate residues participate in the catalytic mechanism (1, 2). In analogy with the term serine proteases, the term ispartate proteases has been suggested (3).Chymosin is secreted as an inactive precursor, prochymosin, consisting of a single peptide chain with 365 amino acid residues. The zymogen is irreversibly converted into active enzyme by limited proteolysis during which a total of 42 amino acid residues are released from the amino-terminal part of the peptide chain.Prochymosin contains six half-cystine residues forming three disulfide bridges, all located in the enzyme part of the molecule. Investigations on the primary structure began with determination of the amino acid sequences around the disulfide bridges (4). In subsequent papers (5, 6) we reported the sequence of the activation segment and the sequence of the first 61 residues of the active enzyme.This communication presents the complete amino acid sequence of prochymosin B. It is shown that this zymogen is highly homologous to porcine pepsinogen A (7-10). To facilitate the comparison, we have chosen to number the amino acid residues from the NH2-terminus of prochymosin and then continue by counting gaps where such occur in prochymosin relative to pepsinogen. The comparison is further extended to penicillopepsin, the only other aspartate protease of which the sequence is almost completely known (11). METHODSProchymosin and chymosin used in these investigations were prepared in our laboratory according to the methods described previously (12).The sequence was obtained after a series of degradation experiments carried out in parallel. In most cases the first steps were enzymatic cleavage with trypsin or chemical cleavage with cyanogen bromide. To improve solubility of the digest and to reduce the number of fragments, we used maleylated or citraconylated preparations for the digestions with trypsin. The digestions were carried out at 120 (pH 8, for 15-30 order to avoid unspecific, chymotrypsin-like cleavages (13). After such treatment the large fragments were purified by gel filtration on Sephadex G-100 in 0.05 M NH4HCO3, pH 8, with 8 M urea. After cleavage of chymosin with cyanogen bromide the fragments were purified by gel filtration on Sephadex G-100 in 25% acetic acid. The best results were obtained if cleavage was performed on enzyme with inta...

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By conversion of prochymosin into active chymosin an N‐terminal segment of 42 amino acid residues is liberated. In one activation experiment this segment was recovered in two peptides; in a second experiment the activation segment was cleaved into three peptides. The primary structures of the peptides have been determined. Overlaps between these peptides and between the activation segment and the active enzyme have been obtained from peptides produced by tryptic digestion of denatured prochymosin. Comparison of the amino acid sequences of the activation segments from bovine prochymosin, bovine pepsinogen and porcine pepsinogen shows considerable homology.

Activation of prochymosin at pH below 2.5 results in formation of the active enzyme pseudochymosin by proteolytic cleavage of the bond 27-28. Pseudochymosin is 15 amino acid residues longer than chymosin. It is the final activation product at low pH, whereas chymosin is formed by activation between pH 4 and 5. Pseudochymosin is converted to chymosin when it is brought to pH 5.5.Our present knowledge does not allow quantitative evaluation of the possible reactions involved in formation of pseudochymosin, but the course of activation at pH 2 is in accordance with an intermolecular reaction between two zymogen molecules as the predominant reaction. We find indications of an intramolecular reaction when intermolecular reactions are prevented by immobilization of the zymogen.