Folding strategy to prepare Co(II)-substituted metallo-beta-lactamase L1.
In an effort to overcome previous problems with the preparation of Co(II)-substituted metallo-beta-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 k(cat) of 63 s(-1) and K(m) of 20 microM 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).
Hu
Zhenxin
Z
Department of Chemistry and Biochemistry, 160 Hughes Hall, Miami University, Oxford, OH 45056, USA.
Periyannan
Gopal R
GR
Crowder
Michael W
MW
D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D003035:Cobalt / Q000378:metabolism*; D007700:Kinetics; D010720:Phosphines / Q000494:pharmacology; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D015032:Zinc; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism*
D001051:Apoenzymes; D003035:Cobalt; D010720:Phosphines; D015032:Zinc; D001618:beta-Lactamases; C080938:tris(2-carboxyethyl)phosphine; C088032:beta-lactamase L1