Site-directed mutagenesis and molecular modelling studies show the role of Asp82 and cysteines in rat acylase 1, a member of the M20 family.
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.
Herga
Sameh
S
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-Jérôme, 13397 Marseille Cedex 20, France.
Brutus
Alexandre
A
Vitale
Rosa Maria
RM
Miche
Hélène
H
Perrier
Josette
J
Puigserver
Antoine
A
Scaloni
Andrea
A
Giardina
Thierry
T
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry*; D001483:Base Sequence; D003545:Cysteine / Q000737:chemistry*; D017931:DNA Primers; D007668:Kidney / Q000201:enzymology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D051381:Rats
D017931:DNA Primers; D001224:Aspartic Acid; D000581:Amidohydrolases; C019665:amidase; D003545:Cysteine