The problem of a solvent exposable disulfide when preparing Co(II)-substituted metallo-β-lactamase L1 from Stenotrophomonas maltophilia

摘要

In an effort to prepare Co(II)-substituted metallo-β-lactamase L1 from Stenotrophomonas maltophilia for future spectroscopic and mechanistic studies, two methods for the preparation of Co(II)-L1 were tested. Method A involved adding CoCl_2 directly to apo-L1 under anaerobic conditions. The resulting enzyme contained 1.9 moles of cobalt and exhibited very little activity, and UV-Vis, ~1H NMR, and EPR studies indicated that most of the cobalt in this sample was Co(III), Method B involved reducing the single and unique disulfide bridge in L1 with tris(carboxyethyl)phosphine prior to adding CoCl_2. The resulting enzyme was pink, contained 2.5 moles of cobalt per mole of enzyme, and exhibited K_(cat) and K_m values of 18 ± 1 s~(-1) and 10 ± 1 μM, respectively, when using nitrocefin as the substrate. UV-Vis, ~1H NMR, and EPR studies revealed that this enzyme sample contained high-spin Co(II). The UV-Vis spectra also provided evidence for Co(II) bound to one or both of the reduced cysteines. Efforts to block this non-specific Co(II) binding site using a chemical modification agent or site-directed mutagenesis were unsuccessful. The data presented here demonstrate the problem of solvent-exposed disulfides when preparing Co(II)-substituted enzymes and offers a convenient method to circumvent the problem.