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J. Biol. Chem., Vol. 281, Issue 27, 18581-18590, July 7, 2006

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Homology Modeling and Site-directed Mutagenesis of Pyroglutamyl Peptidase II

INSIGHTS INTO OMEGA-VERSUS AMINOPEPTIDASE SPECIFICITY IN THE M1 FAMILY^*

Lucía Chávez-Gutiérrez, Edna Matta-Camacho, Joel Osuna, Eduardo Horjales^¶, Patricia Joseph-Bravo, Bernard Maigret^||, and Jean-Louis Charli¹

From the Departamento de Genética del Desarrollo y Fisiología Molecular, Departamento de Ingeniería Celular y Biocatálisis, and ^¶Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, 62271 Cuernavaca, Morelos, México and ^||Equipe de Dynamique des Assemblages Membranaires, UMR7565, CNRS/Université Henri Poincaré, Nancy 7565, France

Pyroglutamyl peptidase II (PPII), a highly specific membrane-bound omegapeptidase, removes N-terminal pyroglutamyl from thyrotropin-releasing hormone (<Glu-His-Pro-NH₂), inactivating the peptide in the extracellular space. PPII and enzymes with distinct specificities such as neutral aminopeptidase (APN), belong to the M1 metallopeptidase family. M1 aminopeptidases recognize the N-terminal amino group of substrates or inhibitors through hydrogen-bonding to two conserved residues (Gln-213 and exopeptidase motif Glu-355 in human APN), whereas interactions involved in recognition of pyroglutamyl residue by PPII are unknown. In rat PPII, the conserved exopeptidase residue is Glu-408, whereas the other one is Ser-269. Given that variations in M1 peptidase specificity are likely due to changes in the catalytic region, we constructed three-dimensional models for the catalytic domains of PPII and APN. The models showed a salt bridge interaction between PPII-Glu-408 and PPII-Lys-463, whereas the equivalent APN-Glu-355 did not participate in a salt bridge. Docking of thyrotropin-releasing hormone in PPII model suggested that the pyroglutamyl residue interacted with PPII-Ser-269. According to our models, PPII-S269Q and -K463N mutations should leave Glu-408 in a physicochemical context similar to that found in M1 aminopeptidases; alternatively, PPII-S269E replacement might be sufficient to transform PPII into an aminopeptidase. These hypotheses were supported by site-directed mutagenesis; the mutants lost omegapeptidase but displayed alanyl-aminopeptidase activity. In conclusion, recognition of a substrate without an N-terminal charge requires neutralization of the aminopeptidase anionic binding site; furthermore, shortening of side chain at PPII-269 position is required for adjustment to the pyroglutamyl residue.

Received for publication, February 14, 2006 , and in revised form, April 10, 2006.

^* This work was supported by Consejo Nacional de Ciencia y Tecnologia Grant 39931 (to J.-L. C.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2 and Table 1.

¹ To whom correspondence should be addressed: Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Cuernavaca, Mor., 62271, México. Tel.: 52-5556227633; Fax: 52-5556227622; E-mail: charli{at}ibt.unam.mx.

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