Título

Biochemical characterization and structural prediction of a novel cytosolic leucyl aminopeptidase of the M17 family from schizosaccharomyces pombe

Autor

ARTURO ROJO DOMINGUEZ

IRMA PILAR HERRERA CAMACHO

NORA HILDA ROSAS MURRIETA

Nivel de Acceso

Acceso Abierto

Licencia

http://creativecommons.org/licenses/by-nc-nd/4.0

Referencia de datos

doi: 10.1111/j.1742-4658.2007.06142.x

Materias

MICROBIOLOGÍA - (CTI) Mecanismo Catalítico LAP yspII Aminopeptidasa Familia M17 de Peptidasas

Resumen o descripción

A new leucyl aminopeptidase activity has been identified in the fission yeast Schizosaccharomyces pombe. The enzyme, which has been purified and named leucyl aminopeptidase yspII (LAP yspII), had a molecular mass of 320 and 54 kDa by gel filtration and SDS⁄PAGE, respectively, suggesting a homohexameric structure. The enzyme cleaved synthetic aminoacyl-4- nitroanilides at an optimum of pH 8.5, and preferred leucine and methionine as N-terminal amino acids. A clear dependence on Mn2+ concentration for activity was found, and an apparent association constant of 0.33 mm was calculated for the metal ion. Bestatin behaved as a competitive inhibitor of LAP yspII (Ki ¼ 0.14 lm), while chelating agents such as chloroquine, EDTA and 1,10-phenanthroline also reduced enzyme activity. A MALDIMS analysis, followed by sequencing of two of the resulting peptides, showed that LAP yspII undoubtedly corresponds to the putative aminopeptidase C13A11.05 identified in the S. pombe genome project. The protein exhibited nearly 40% sequence identity to fungal and mammalian aminopeptidases belonging to the M17 family of metallopeptidases. Catalytic residues (Lys292 and Arg366), as well as those involved in coordination with the cocatalytic metal ions (Lys280, Asp285, Asp303, Asp362 and Glu364) and those forming the hydrophobic pocket for substrate binding (Met300, Asn360, Ala363, Thr390, Leu391, Ala483 and Met486), were perfectly conserved among all known aminopeptidases. The S. pombe enzyme is predicted to be formed two clearly distinguished domains with a well conserved C-terminal catalytic domain showing a characteristic topology of eight b-sheets surrounded by a-helical segments in the form of a saddle.

Editor

FEBS Journal

Fecha de publicación

2007

Tipo de publicación

Artículo

Recurso de información

http://ilitia.cua.uam.mx:8080/jspui/handle/123456789/219

Formato

application/pdf

Idioma

Inglés

Repositorio Orígen

Concentración de Recursos de Información Científica y Académica, UAM Cuajimalpa

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