Differential Binding of Co(II) and Zn(II) to Metallo-β-Lactamase Bla2 from Bacillus anthracis
Format of Original
American Chemical Society
Journal of the American Chemical Society
Original Item ID
In an effort to probe the structure, mechanism, and biochemical properties of metallo-β-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, 1H 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(II)-substituted proteins to the native Zn(II)-containing forms.
Hawk, Megan J.; Breece, Robert M.; Hajdin, Christine E.; Bender, Katherine M.; Hu, Zhenxin; Costello, Alison L.; Bennett, Brian; Tierney, David L.; and Crowder, Michael W., "Differential Binding of Co(II) and Zn(II) to Metallo-β-Lactamase Bla2 from Bacillus anthracis" (2009). Physics Faculty Research and Publications. 16.