Article (Scientific journals)
Role of Cys221 and Asn116 in the zinc-binding sites of the Aeromonas hydrophila metallo-beta-lactamase.
Vanhove, Marc; Zakhem, M.; Devreese, B. et al.
2003In Cellular and Molecular Life Sciences, 60 (11), p. 2501-9
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Keywords :
Aeromonas hydrophila/enzymology; Asparagine; Bacterial Proteins/chemistry/metabolism; Base Sequence; Binding Sites; Cysteine; Kinetics; Molecular Sequence Data; Structure-Activity Relationship; Zinc/metabolism; beta-Lactamases/chemistry/metabolism
Abstract :
[en] The CphA metallo-beta-lactamase produced by Aeromonas hydrophila exhibits two zinc-binding sites. Maximum activity is obtained upon binding of one zinc ion, whereas binding of the second zinc ion results in a drastic decrease in the hydrolytic activity. In this study, we analyzed the role of Asn116 and Cys221, two residues of the active site. These residues were replaced by site-directed mutagenesis and the different mutants were characterized. The C221S and C221A mutants were seriously impaired in their ability to bind the first, catalytic zinc ion and were nearly completely inactive, indicating a major role for Cys221 in the binding of the catalytic metal ion. By contrast, the binding of the second zinc ion was only slightly affected, at least for the C221S mutant. Mutation of Asn116 did not lead to a drastic decrease in the hydrolytic activity, indicating that this residue does not play a key role in the catalytic mechanism. However, the substitution of Asn116 by a Cys or His residue resulted in an approximately fivefold increase in the affinity for the second, inhibitory zinc ion. Together, these data suggested that the first zinc ion is located in the binding site involving the Cys221 and that the second zinc ion binds in the binding site involving Asn116 and, presumably, His118 and His196.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Vanhove, Marc
Zakhem, M.
Devreese, B.
Franceschini, N.
Anne, Christine ;  Université de Liège - ULiège > Centre d'ingénierie des protéines
Bebrone, Carine ;  Université de Liège - ULiège > Centre d'ingénierie des protéines
Amicosante, G.
Rossolini, G. M.
Van Beeumen, J.
Frère, Jean-Marie ;  Université de Liège - ULiège > Département des sciences de la vie > Département des sciences de la vie
Galleni, Moreno ;  Université de Liège - ULiège > Département des sciences de la vie > Macromolécules biologiques
Language :
English
Title :
Role of Cys221 and Asn116 in the zinc-binding sites of the Aeromonas hydrophila metallo-beta-lactamase.
Publication date :
2003
Journal title :
Cellular and Molecular Life Sciences
ISSN :
1420-682X
eISSN :
1420-9071
Publisher :
Birkhauser Verlag, Switzerland
Volume :
60
Issue :
11
Pages :
2501-9
Peer reviewed :
Peer Reviewed verified by ORBi
Commentary :
The authors acknowledge Springer Verlag. The original publication is available at http://springerlink.com/content/h6elkpyfdlcp4jq4/
Available on ORBi :
since 15 January 2010

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