Influence of charge distribution at the active site surface on the substrate specificity of human neutrophil protease 3 and elastase. A kinetic and molecular modeling analysis.
The biological functions of human neutrophil protease 3 (Pr3) differ from those of neutrophil elastase despite their close structural and functional resemblance. Although both proteases are strongly cationic, their sequences differ mainly in the distribution of charged residues. We have used these differences in electrostatic surface potential in the vicinity of their active site to produce fluorescence resonance energy transfer (FRET) peptide substrates for investigating individual Pr3 subsites. The specificities of subsites S5 to S3' were investigated both kinetically and by molecular dynamic simulations. Subsites S2, S1', and S2' were the main definers of Pr3 specificity. Combinations of results for each subsite were used to deduce a consensus sequence that was complementary to the extended Pr3 active site and was not recognized by elastase. Similar sequences were identified in natural protein substrates such as NFkappaB and p21 that are specifically cleaved by Pr3. FRET peptides derived from these natural sequences were specifically hydrolyzed by Pr3 with specificity constants k(cat)/K(m) in the 10(6) m(-1) s(-1) range. The consensus Pr3 sequence may also be used to predict cleavage sites within putative protein targets like the proform of interleukin-18, or to develop specific Pr3 peptide-derived inhibitors, because none is available for further studies on the physiopathological function of this protease.
Korkmaz
Brice
B
INSERM U618, Faculty of Medicine, 10 Bd. Tonnellé, 37032 Tours Cedex, France.
Hajjar
Eric
E
Kalupov
Timofey
T
Reuter
Nathalie
N
Brillard-Bourdet
Michèle
M
Moreau
Thierry
T
Juliano
Luiz
L
Gauthier
Francis
F
D000595:Amino Acid Sequence; D001665:Binding Sites; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D020382:Interleukin-18 / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D053146:Myeloblastin / Q000737:chemistry*; D016328:NF-kappa B / Q000378:metabolism; D010196:Pancreatic Elastase / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D013379:Substrate Specificity
D050759:Cyclin-Dependent Kinase Inhibitor p21; D020382:Interleukin-18; D016328:NF-kappa B; D010196:Pancreatic Elastase; D053146:Myeloblastin