Use of a 49-peptide library for a qualitative and quantitative determination of pseudomonal serralysin specificity

The Pseudomonas aeruginosa serralysin (E.C. 3.4.24.40.), which is a zinc-dependent metalloprotease from the metzincin superfamily, has quite a broad specificity, which has not yet been clearly identified. We have studied it with an original approach, using a 49-peptide library of the type Z-AXXA (amide) (X = A, L, V, F, S, R, E). The library was analyzed by LC-MS before: and after enzymatic hydrolysis. A great number of hydrolyzed peptides were screened and the preferential hydrolysis was the X-X peptide bond, even if in some cases, A-X and X-A bond could be hydrolyzed. No amino acids with a ionized side chain could be found in the P-1' position. The results obtained suggest that the specificity in the P-n' position, where an hydrophobic residue was preferentially found, seems more selective that in the P-n position. The P-1 position was not very specific, but, on a quantitative point of view, the enzymatic activity was particularly increased when R, F or A were in this position. The results allow us to define the P-1' and P-1 residues for an optimal substrate of pseudomonal serralysin and usable for the design and the synthesis of a specific inhibitor. (C) 1999 Elsevier Science Ltd. All rights reserved.