title: Differential binding of Co(II) and Zn(II) to metallo-beta-lactamase Bla2 from Bacillus anthracis.
abstract: In an effort to probe the structure, mechanism, and biochemical properties of metallo-beta-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, (1)H 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.
authors: Department of Chemistry and Biochemistry, 160 Hughes Hall, Miami University, Oxford, Ohio 45056, USA.
authors: Megan J
authors: MJ
authors: Hawk
authors: Robert M
authors: RM
authors: Breece
authors: Christine E
authors: CE
authors: Hajdin
authors: Katherine M
authors: KM
authors: Bender
authors: Zhenxin
authors: Z
authors: Hu
authors: Alison L
authors: AL
authors: Costello
authors: Brian
authors: B
authors: Bennett
authors: David L
authors: DL
authors: Tierney
authors: Michael W
authors: MW
authors: Crowder
mesh_terms: D001408:Bacillus anthracis / Q000201:enzymology*; D003035:Cobalt / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013057:Spectrum Analysis; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism*; D001618:beta-Lactamases / Q000096:biosynthesis / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D003035:Cobalt; D001618:beta-Lactamases; D015032:Zinc