Site-directed mutagenesis and molecular modelling studies show the role of Asp82 and cysteines in rat acylase 1, a member of the M20 family |
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Authors: | Herga Sameh Brutus Alexandre Vitale Rosa Maria Miche Hélène Perrier Josette Puigserver Antoine Scaloni Andrea Giardina Thierry |
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Institution: | Institut Méditerranéen de Recherche en Nutrition, Laboratoire de Biochimie et Biologie de la Nutrition, UMR Université Paul Cézanne Aix Marseille III-INRA 1111, service 342, Faculté des Sciences et Techniques Saint-Jér?me, 13397 Marseille Cedex 20, France. |
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Abstract: | Acylase 1 from rat kidney catalyzes the hydrolysis of acyl-amino acids. Sequence alignment has shown that this enzyme belongs to the metalloprotein family M20. Site-directed mutagenesis experiments led to the identification of one functionally important amino acid residue located near one of the zinc coordinating residues, which play a critical role in the enzymatic activity. The D82N- and D82E-substituted forms showed no significant activity and very low activity, respectively, along with a loss of zinc coordination. Molecular modelling investigations indicated a putative role of D82 in ensuring a proper protonation of catalytic histidine. In addition, none of the five cysteine residues present in the rat kidney acylase 1 sequence seemed involved in the catalytic process: the loss of activity induced by the C294A substitution was probably due to a conformational change in the 3D structure. |
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Keywords: | Site-directed mutagenesis Acylase I Molecular modelling M20 family |
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