Folding strategy to prepare Co(II)-substituted metallo-beta-lactamase L1 |
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Authors: | Hu Zhenxin Periyannan Gopal R Crowder Michael W |
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Institution: | Department of Chemistry and Biochemistry, 160 Hughes Hall, Miami University, Oxford, OH 45056, USA |
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Abstract: | In an effort to overcome previous problems with the preparation of Co(II)-substituted metallo-β-lactamase L1, two strategies were undertaken. Attempts to prepare Co(II)-substituted L1 using biological incorporation resulted in an enzyme that contained only 1 Eq of cobalt and exhibited no catalytic activity. Co(II)-substituted L1 could be prepared by refolding metal-free L1 in the presence of Co(II), and the resulting enzyme contained 1.8 Eq of cobalt, yielded a UV-Vis spectrum consistent with 5-coordinate Co(II), and exhibited a kcat of 63 s−1 and Km of 20 μM when using nitrocefin as the substrate. Pre-steady-state fluorescence and UV-Vis studies demonstrated that refolded, Co(II)-substituted L1 uses the same kinetic mechanism as Zn(II)-containing L1, in which a reaction intermediate is formed when using nitrocefin as substrate. The described refolding strategy can be used to prepare other Co(II)-substituted Zn(II)-metalloenzymes, particularly those that contain a solvent-exposable disulfide, which often causes oxidation of Co(II) to Co(III). |
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Keywords: | Co(II) substitution Zn(II)-metalloenzyme Metallo-β-lactamase Refolding |
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