Practical Protein Chemistry - A. Darbre 1989

Enzymatic fragmentation of the polypeptide chain
Proteases with high specificity
Protease from Armillaria mellea

The enzyme was isolated from the basidiomycete Armillaria mellea [110] and obtained as an active preparation [60, 110, 111]. It has been shown to be a metalloprotein (containing Zn²⁺) with a Molecular Weight of 14,000 and a pH optimum of 6.8. The enzyme is not commercially available.

3.5.6.1. Specificity and Hydrolysis conditions.

Specificity. Studies on the enzyme's specificity revealed that hydrolysis occurs at the N-terminal peptide bond of Lysine and aminoethylcysteine (Aec) residues, specifically at -X-Lys- and -X-Aec- bonds, even when X = Pro. Cleavage of the -Pro-Lys bond was observed during the Digestion of Insulin and other substrates [16, 36]. It was also demonstrated that peptide bonds featuring acidic residues adjacent to lysine are resistant to the enzyme's action. In some cases, hydrolysis of the C-terminal peptide bond of Arginine (-Arg-X) is observed. The extent of hydrolysis at this bond increases when X = Leu or Ile, although not all fragments of this type undergo hydrolysis.

The enzyme from A. mellea has been utilized for the Limited proteolysis of IMMUNOGLOBULINS G and D [44, 48]. The results of these studies indicate its high specificity.

Hydrolysis conditions. Hydrolysis is carried out in 0.2 M N-ethylmorpholine-acetate buffer (pH 8.0) at an Enzyme-to-substrate ratio of 1:100 at 35 °C for 22 h [15], or in 0.1 M NH₄HCO₃ at an enzyme-to-substrate ratio of 1:500–1000 at 37 °C [36]. Satisfactory results are obtained when the reaction is performed within a pH range of 4–9 and at temperatures elevated up to 50 °C. Optimal results are achieved by treating substrates in 10 mM Tris-HCl buffer (pH 7.0) containing 0.15 M NaCl and 2 mM calcium acetate, at an enzyme-to-substrate ratio of 1:100 at 37 °C for 3 h [44]. The enzyme is inhibited in the presence of EDTA, as well as by 1 mM solutions of lysine or Cysteine [111].

Specific Cleavage at cysteine residues. Using the human Hemoglobin ß-chain and bee venom toxin as Examples, it was demonstrated that the enzyme from A. mellea can be employed for the specific hydrolysis of aminoethylcysteine residues [15]. Following the Modification of lysine residues via trifluoroacetylation, the specified Proteins were alkylated at their SH-groups using ethylenimine. Enzyme Treatment resulted in specific hydrolysis at all aminoethylcysteine residues, with the exception of -Aec-Lys- and -Lys-Aec- fragments.