Inspection of the Binding Sites of Proteinase3 for the Design of a Highly Specific Substrate

Abstract

Proteinase3 (PR3) and human neutrophil elastase (HNE) are homologous proteases from the polymorphonuclear neutrophils and have been thought for a long time to have close enzymatic specificity. We have used molecular dynamics simulations to investigate and compare the interactions between different peptides and the two enzymes. The important role played especially by the C-terminal part of the peptides is confirmed. We provide a map of the subsites of PR3 and a description of the interaction scheme for six ligands. The main difference between HNE and PR3 concerns S2, S1', S2', and S3'. The recognition subsites in PR3 are interconnected; in particular, Lys99 participates to a hydrophobic (S4) and a polar (S2) pocket. On the basis of the simulations, we suggest that VADVKDR is a highly specific sequence for PR3; enzymatic assays confirm that it is cleaved by PR3 with a high specificity constant (k(cat)/K(m) = 3,400,000 M(-1) s(-1)) and not by HNE.

Neutrophil-Derived Proteinase 3 Induces Kallikrein-Independent Release of a Novel Vasoactive Kinin

“…Although this represents the ideal sequence, other amino acids may be found at the cleavage sites (44,45). The cleavage sites on HK do not fully match the consensus sequence established for PR3 (46) (Fig.…”

“…To determine the optimal time for incubation of PR3 with purified HK, purified HK (5 g/ml) was incubated with PR3 (1 and 10 g/ml) for 1,5,10,20,30,45,60, and 90 min. Liberation of kinins peaked at 20 min and declined toward 90 min.…”

Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases

“…Indeed, most commercially available chromogenic and/or fluorogenic substrates used to measure HNE and CG display a proline at the P2 position. Because of the presence of Lys99, PR3 preferentially accommodates a negatively charged residue at P2 (Hajjar et al, 2006;Korkmaz et al, 2007). The S3 subsite of CG is formed by a Lys at position 192, which favors interaction with an acidic P3 residue (Tanaka et al, 1985).…”

Inhibitors and Antibody Fragments as Potential Anti-Inflammatory Therapeutics Targeting Neutrophil Proteinase 3 in Human Disease

“…Molecular dynamics simulations based on the threedimensional structure reveal that Asp61 in PR3 is close to the putative subsites S19 and S39, and Arg143 contributes to the shape of the S29 pocket (Hajjar et al, 2006;Korkmaz et al, 2007). The 60 loop containing Asp61 is significantly displaced to bring the negatively charged side chain close to the S19 and S39 sites.…”

“…By contrast, the Lys/Leu substitution at position 99 makes the S2 subsite of HNE quite hydrophobic. PR3 preferentially accommodates a negatively charged (Asp, Glu) or a hydrophilic residue (Tyr, Ser) at P2 because of its Lys99 (Hajjar et al, 2006;Korkmaz et al, 2007Korkmaz et al, , 2013a. The replacement of Lys99 by Leu in a recently described recombinant PR3 mutant (PR3K99L) considerably reduced the rate at which PR3 substrates were hydrolyzed (Jégot et al, 2011).…”