[en] The role of the mobile loop comprising residues 60-66 in metallo-beta-lactamases has been studied by site-directed mutagenesis, determination of kinetic parameters for six substrates and two inhibitors, pre-steady-state characterization of the interaction with chromogenic nitrocefin, and molecular modeling. The W64A mutation was performed in IMP-1 and BcII (after replacement of the Bcll 60-66 peptide by that of IMP-1) and always resulted in increased K-i and K. and decreased k(cat)/K-m values, an effect reinforced by complete deletion of the loop. k(cat) values were, by contrast, much more diversely affected, indicating that the loop does not systematically favor the best relative positioning of substrate and enzyme catalytic groups. The hydrophobic nature of the ligand is also crucial to strong interactions with the loop, since imipenem was almost insensitive to loop modifications.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Moali, Catherine; Université de Liège - ULiège > Centres généraux > Centre d'ingénierie des protéines
Anne, Christine ; Université de Liège - ULiège > Centres généraux > Centre d'ingénierie des protéines
Lamotte-Brasseur, Josette ; Université de Liège - ULiège > Centres généraux > Centre d'ingénierie des protéines
Groslambert, Sylvie ; Université de Liège - ULiège > Department of Chemical Engineering > PEPs (Product, Environment, Processes)
Devreese, B.
Van Beeumen, J.
Galleni, Moreno ; Université de Liège - ULiège > Département des sciences de la vie > Macromolécules biologiques
Frère, Jean-Marie ; Université de Liège - ULiège > Département des sciences de la vie > Département des sciences de la vie
Language :
English
Title :
Analysis of the importance of the metallo-beta-lactamase active site loop in substrate binding and catalysis
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