Differential Binding of Co(II) and Zn(II) to Metallo-β-Lactamase Bla2 from Bacillus anthracis
Differential Binding of Co(II) and Zn(II) to Metallo-β-Lactamase Bla2 from Bacillus anthracis
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DOI：
10.1021/ja900296u
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发表时间：
2009-08-05
影响因子：
15
通讯作者：
Crowder, Michael W.
中科院分区：
文献类型：
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作者：
Hawk, Megan J.;Breece, Robert M.;Crowder, Michael W.
为了探索炭疽芽孢杆菌金属β-内酰胺酶Bla2的结构、作用机制和生化性质，对该酶进行了高效表达、纯化和性质研究。金属分析表明，重组BLA2与1当量的锌(II)紧密结合。稳态动力学研究表明，单锌(II)BA12是活性的，而双锌(II)BA2是不稳定的。在催化方面，1Zn-Bla2的行为类似于相关的酶CCRA和L1。相反，二钴(II)BA12(Coco-BLA2)比单钴(II)类似物更具活性。快速动力学和UV-Vis、H-1核磁共振、EPR和EXAFS光谱研究表明，Co(II)与Bla2的结合是分布的，而EXAFS则表明锌(II)的结合是顺序的。据我们所知，这是第一个有文献记载的锌酶通过不同的机制与钴(II)和锌(II)结合的例子，强调了在将Co(Il)取代的蛋白质的结果外推到天然含锌(II)的形式时证明可转移性的必要性。
In an effort to probe the structure, mechanism, and biochemical properties of metallo-p-lactamase Bla2 from Bacillus anthracis, the enzyme was overexpressed, purified, and characterized. Metal analyses demonstrated that recombinant Bla2 tightly binds 1 equiv of Zn(II). Steady-state kinetic studies showed that mono-Zn(II) Bla2 (1Zn-Bla2) is active, while di-Zn(II) Bla2 (ZnZn-Bla2) was unstable. Catalytically, 1Zn-Bla2 behaves like the related enzymes CcrA and L1. In contrast, di-Co(II) Bla2 (CoCo-Bla2) is substantially more active than the mono-Co(II) analogue. Rapid kinetics and UV-vis, H-1 NMR, EPR, and EXAFS spectroscopic studies show that Co(II) binding to Bla2 is distributed, while EXAFS shows that Zn(II) binding is sequential. To our knowledge, this is the first documented example of a Zn enzyme that binds Co(II) and Zn(II) via distinct mechanisms, underscoring the need to demonstrate transferability when extrapolating results on Co(Il)-substituted proteins to the native Zn(II)-containing forms.