Paul-Soto R, Zeppezauer M, Adolph H W, Galleni M, Frere J M, Carfi A, Dideberg O, Wouters J, Hemmingsen L, Bauer R
Department of Physics, The Royal Veterinary and Agricultural University, Frederiksberg C, Denmark.
Biochemistry. 1999 Dec 14;38(50):16500-6. doi: 10.1021/bi9911381.
Cd-substituted forms of the Bacillus cereus metallo-beta-lactamases (BCII) were studied by perturbed angular correlation of gamma-rays (PAC) spectroscopy. At very low [Cd]:[apo-beta-lactamase] ratios, two nuclear quadrupole interactions (NQI) were detected. For [Cd]:[apo-beta-lactamase] ratios between 0.8 and 3.0, two new NQIs appear, and the spectra show that up to 2 cadmium ions can be bound per molecule of apoenzyme. These results show the existence of two interacting Cd-binding sites in BCII. The relative populations of the two NQIs found at low [Cd]:[apo-beta-lactamase] ratios yielded a 1:3 ratio for the microscopic dissociation constants of the two different metal sites (when only one cadmium ion is bound). X-ray diffraction data at pH 7.5 demonstrate that also for Zn(II) two binding sites exist, which may be bridged by a solvent molecule. The measured NQIs could be assigned to the site with three histidines as metal ligands (three-His site) and to the site with histidine, cysteine, and aspartic acid as metal ligands (Cys site), respectively, by PAC measurements on the Cys168Ala mutant enzyme. This assignment shows that cadmium ions preferentially bind to the Cys site. This is in contrast to the preference of Zn(II) in the hybrid Zn(II)Cd(II) enzyme, where an analysis of the corresponding PAC spectrum showed that Cd(II) occupied the Cys site, whereby Zn(II) occupied the site with three histidines. The difference between Zn(II) and Cd(II) in affinity for the two sites is combined with the kinetics of hydrolysis of nitrocefin for different metal ion substitutions (Zn(2)E, ZnE, Cd(2)E, CdE, and ZnCdE) to study the function of the two metal ion binding sites.
通过γ射线扰动角关联（PAC）光谱研究了蜡样芽孢杆菌金属β-内酰胺酶（BCII）的镉取代形式。在非常低的[Cd]：[脱辅基β-内酰胺酶]比率下，检测到两种核四极相互作用（NQI）。对于[Cd]：[脱辅基β-内酰胺酶]比率在0.8至3.0之间，出现了两种新的NQI，并且光谱表明每分子脱辅基酶最多可结合2个镉离子。这些结果表明BCII中存在两个相互作用的镉结合位点。在低[Cd]：[脱辅基β-内酰胺酶]比率下发现的两种NQI的相对丰度，对于两个不同金属位点的微观解离常数产生了1：3的比率（当仅结合一个镉离子时）。pH 7.5下的X射线衍射数据表明，对于锌（II）也存在两个结合位点，这两个位点可能由一个溶剂分子桥接。通过对Cys168Ala突变体酶进行PAC测量，测得的NQI可分别归属于以三个组氨酸作为金属配体（三个组氨酸位点）的位点和以组氨酸、半胱氨酸和天冬氨酸作为金属配体（半胱氨酸位点）的位点。该归属表明镉离子优先结合到半胱氨酸位点。这与杂合锌（II）镉（II）酶中锌（II）的偏好相反，对相应PAC光谱的分析表明，镉（II）占据半胱氨酸位点，而锌（II）占据以三个组氨酸的位点。锌（II）和镉（II）对两个位点亲和力的差异与不同金属离子取代（Zn(2)E、ZnE、Cd(2)E、CdE和ZnCdE）时硝基头孢菌素的水解动力学相结合，以研究两个金属离子结合位点的功能。
