The problem of a solvent exposable disulfide when preparing Co(II)-substituted metallo-beta-lactamase L1 from Stenotrophomonas maltophilia.
In an effort to prepare Co(II)-substituted metallo-beta-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 CoCl2 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 CoCl2. The resulting enzyme was pink, contained 2.5 moles of cobalt per mole of enzyme, and exhibited kcat and Km values of 18+1 s(-1) and 10+/-1 microM, 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.
Crowder
M W
MW
Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA. crowdemw@muohio.edu
Yang
K W
KW
Carenbauer
A L
AL
Periyannan
G
G
Seifert
M E
ME
Rude
N E
NE
Walsh
T R
TR
D001483:Base Sequence; D003035:Cobalt / Q000737:chemistry*; D017931:DNA Primers; D004220:Disulfides / Q000737:chemistry*; D008958:Models, Molecular; D012997:Solvents / Q000737:chemistry; D013057:Spectrum Analysis; D020588:Stenotrophomonas / Q000201:enzymology*; D001618:beta-Lactamases / Q000737:chemistry*
D017931:DNA Primers; D004220:Disulfides; D012997:Solvents; D003035:Cobalt; C088032:beta-lactamase L1; D001618:beta-Lactamases