Metal ion binding and coordination geometry for wild type and mutants of metallo-beta -lactamase from Bacillus cereus 569/H/9 (BcII): a combined thermodynamic, kinetic, and spectroscopic approach.
One high affinity (nm) and one low affinity (microM) macroscopic dissociation constant for the binding of metal ions were found for the wild-type metallo-beta-lactamase from Bacillus cereus as well as six single-site mutants in which all ligands in the two metal binding sites were altered. Surprisingly, the mutations did not cause a specific alteration of the affinity of metal ions for the sole modified binding site as determined by extended x-ray absorption fine structure (EXAFS) and perturbed angular correlation of gamma-rays spectroscopy, respectively. Also UV-visible absorption spectra for the mono-cobalt enzymes clearly contain contributions from both metal sites. The observations of the very similar microscopic dissociation constants of both binding sites in contrast to the significantly differing macroscopic dissociation constants inevitably led to the conclusion that binding to the two metal sites exhibits negative cooperativity. The slow association rates for forming the binuclear enzyme determined by stopped-flow fluorescence measurements suggested that fast metal exchange between the two sites for the mononuclear enzyme hinders the binding of a second metal ion. EXAFS spectroscopy of the mono- and di-zinc wild type enzymes and two di-zinc mutants provide a definition of the metal ion environments, which is compared with the available x-ray crystallographic data.
de Seny
D
D
Centre d'Ingéniérie des Protéines, Institut de Chimie B6, Université de Liège, Sart-Tilman, B-4000 Liège, Belgium.
Heinz
U
U
Wommer
S
S
Kiefer
M
M
Meyer-Klaucke
W
W
Galleni
M
M
Frere
J M
JM
Bauer
R
R
Adolph
H W
HW
D001409:Bacillus cereus / Q000201:enzymology*; D001665:Binding Sites; D001667:Binding, Competitive; D002104:Cadmium / Q000378:metabolism; D018360:Crystallography, X-Ray; D019992:Databases as Topic; D005720:Gamma Rays; D007477:Ions; D007700:Kinetics; D008024:Ligands; D008670:Metals / Q000494:pharmacology; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013379:Substrate Specificity; D014466:Ultraviolet Rays; D014965:X-Rays; D015032:Zinc / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism*
D007477:Ions; D008024:Ligands; D008670:Metals; D002104:Cadmium; D001618:beta-Lactamases; D015032:Zinc