Alpha-Lytic Protease M190A Mutant

Alpha-lytic protease (aLP) is an alternative specificity protease for proteomics applications, whose wild-type (WT) version cleaves after T, A, S, and V residues. The M190A (Met190 → Ala190) mutant of aLP has different cleavage specificities, and cleaves after M, F, and L residues. Both the WT and M190A forms of aLP generate peptides of similar average length as trypsin.

In WT aLP, the methionines at positions 190 and 213 cause WT aTP to have its particular specificity toward peptide substrates with small hydrophobic side chains at the P₁ position. The M190A mutation gives the resulting mutant M190A aLP the ability to cleave peptide substrates with large hydrophobic side chains at the P₁ side chain with greater efficiency compared to WT aLP, in addition to accommodating peptides with small hydrophobic side chains as before. Other structural changes in M190A aLP compared to WT aLP have been discussed.

The activity of M190A aLP in the presence of various solution components is as follows:

• 0.1% sodium deoxycholate: ~1.4-fold enhanced activity
• 1.0% sodium deoxycholate: nearly full activity
• 0.1% SDS: ~40% activity
• 1.0% SDS: ~30% activity
• 1 M guanidine HCl: ~20% activity
• 4 M guanidine HCl: ~1% activity (essentially inactivated)

mmols of pNA produced per min per mg protein from 0.5 mM N-succinyl-Ala-Ala-Pro-Phe-PNA at 25 ^°C in 100 mM Tris-HCl (pH 7.5).