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J. Biol. Chem., Vol. 282, Issue 3, 1989-1997, January 19, 2007

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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^*

Brice Korkmaz, Eric Hajjar^¶1, Timofey Kalupov, Nathalie Reuter^¶1, Michèle Brillard-Bourdet, Thierry Moreau, Luiz Juliano^||, and Francis Gauthier²

From the INSERM U618, Faculty of Medicine, 10 Bd. Tonnellé, 37032 Tours Cedex, France, the Université François Rabelais, F37000 Tours, France, the ^¶Computational Biology Unit, Bergen Center for Computational Science, University of Bergen, N-5008 Bergen, Norway, and the ^||Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal, 04044-20 São Paulo, Brazil

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 NFB 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 10⁶ 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.

Received for publication, September 8, 2006 , and in revised form, November 2, 2006.

^* This work was supported in part by the Vaincre la Mucoviscidose and by the Fundação de Amparo a Pesquisa do Estado de São Paulo and Conselho Nacional de Desenvolvimento Científico e Tecnológico. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by the National Program for Research in Functional Genomics in Norway from the Research Council of Norway.

² To whom correspondence should be addressed. Tel.: 33-2-47-36-60-45; Fax: 33-2-47-36-60-46; E-mail: gauthier{at}univ-tours.fr.

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