Sep 23, 1997

Zn(II) dependence of the Aeromonas hydrophila AE036 metallo-beta-lactamase activity and stability

Biochemistry
M Hernandez ValladaresM Galleni

Abstract

Two Zn2+ binding sites were found in the Aeromonas hydrophila AE036 metallo-beta-lactamase. The affinity of the first binding site for Zn2+ ions is so high that the dissociation constant could not be determined, but it is significantly lower than 20 nM. The mono-Zn2+ form of the enzyme exhibits a maximum activity against its carbapenem substrates. The presence of a Zn2+ ion in the second lower affinity binding site results in a loss of enzymatic activity with a Ki value of 46 microM at pH 6.5. The kinetic analysis is in agreement with a noncompetitive inhibition mechanism. The Zn content of the A. hydrophila enzyme is also strongly pH-dependent. With an external Zn2+ ion concentration of 0.4 microM, occupancy of the higher affinity site by metal ions is lower than 10% at pH 5 and 10. The affinity for the second binding site seems to increase from pH 6 to 7.5. Fluorescence emission and circular dichroism spectra revealed slight conformational changes upon titration of the apoenzyme by Zn2+ ions, resulting in the successive saturation of the first and second binding sites. Differential scanning calorimetry transitions and intrinsic fluorescence emission spectra in the presence of increasing concentrations of urea demonstrate that...Continue Reading

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Mentioned in this Paper

Apoenzymes
Aeromonas hydrophila
Fluorescence Spectroscopy
Metalloproteins
Beta-Lactamase
Zinc
Protein Conformation
Circular Dichroism, Vibrational
beta-Lactamase Inhibitors
Metal Antagonists

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