Folding strategy to prepare Co(II)-substituted metallo-β-lactamase L1

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 one equivalent 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 equivalents of cobalt, yielded a UV-Vis spectrum consistent with five-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 utilizes 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).