Practical Protein Chemistry - A. Darbre 1989

Fragmentation of the polypeptide chain by enzymatic methods
Proteases with high specificity
Postproline-specific enzyme

Because the Proline residue is structurally fundamentally different from other amino acid residues while still belonging to the Natural Amino Acids and entering into the composition of many Proteins, one could expect that an enzyme cleaving the corresponding peptide bonds exists in nature. Such an enzyme would be of undoubted interest, since proline is a rare Amino Acid and its presence limits the possibility of attack on neighboring peptide bonds by other Proteolytic Enzymes. To date, only one post-proline-specific enzyme has been isolated [54]. The Isolation and Purification of this enzyme from lamb Kidneys was carried out using Affinity Chromatography. According to preliminary data, the enzyme belongs to the Class of Serine proteases [123]. A detailed characterization of the enzyme is given in [109, 124]. The enzyme is not commercially available.

3.5.8.1. Specificity and Hydrolysis conditions. Using the hydrolysis of short Peptides containing no more than 13 amino acid residues as an example, it was shown that the enzyme hydrolyzes all types of -Pro-X- bonds except for -Pro-Pro- [54]. The rate of hydrolysis depends on The Nature of the X residue: the maximum rate is observed when X has hydrophobic properties, and the minimum when it has acidic or basic properties. It was also shown that the enzyme hydrolyzes -Ala-X- bonds, but the hydrolysis rate is 1/100 to 1/1000 of the rate of -Pro-X- bond hydrolysis. Upon incubation of an octapeptide, limited hydrolysis of the -Pro-Thr- bond was observed [20], whereas Treatment of 22- and 33-membered peptides for 36 h resulted in the Cleavage of only 10–20% of the bonds susceptible to the enzyme's action [23]. The obtained data indicate that the enzyme can be used only for the hydrolysis of short peptides.

Hydrolysis conditions. The enzyme exhibits maximum activity in the pH range of 7.5–8.0. Hydrolysis is carried out in 100 mM NH₄HCO₃ (pH 7.8) containing 1 mM DTT and 1 mM EDTA at 22 °C for 24 h.