Analysis of the importance of the metallo-beta-lactamase active site loop in substrate binding and catalysis.
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 BcII 60-66 peptide by that of IMP-1) and always resulted in increased K(i) and K(m) 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.
Centre d'Ingénierie des Protéines, Université de Liège, Sart-Tilman, B-4000 Liege, Belgium.
Catherine
C
Moali
Christine
C
Anne
Josette
J
Lamotte-Brasseur
Sylvie
S
Groslambert
Bart
B
Devreese
Jozef
J
Van Beeumen
Moreno
M
Galleni
Jean Marie
JM
Frère
D001665:Binding Sites; D002384:Catalysis; D002511:Cephalosporins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019254:Genes, bcl-2; D006868:Hydrolysis; D007202:Indicators and Reagents; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008958:Models, Molecular; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013056:Spectrophotometry, Ultraviolet; D015032:Zinc / Q000737:chemistry; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D002511:Cephalosporins; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D001618:beta-Lactamases; C021720:nitrocefin; D015032:Zinc