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Inhibition of Co (II)‐reconstituted Bacillus cereus 5/B/6 metallo‐β‐lactamase by DNA

Metallo‐β‐lactamase producing strains of pathogenic bacteria have become an increasingly important medical and pharmaceutical problem. The metallo‐β‐lactamase from Bacillus cereus 5/B/6 is capable of catalyzing the hydrolysis of the β‐lactams of a variety of cephalosporin antibiotics including cephalosporin C, cephalexin and cefuroxime. We have previously reported the discovery of a 10‐residue single‐stranded DNA aptamer (ss‐DNA) that is a potent noncompetitive inhibitor of the B. cereus 5/B/6 zinc‐dependent β‐lactamase [R. W. Shaw and S.‐K. Kim, FASEB Journal 17, p. A981 (2003)]. We now report the inhibition of the Co (II)‐reconstituted form of the enzyme by the ss‐DNA. A 250 μM sample of enzyme was incubated in the presence of 25 mM EDTA, pH=7.5 until less than 1% of original activity remained, and then loaded onto a 1.5x50 cm Sephadex G‐25 superfine column previously equilibrated with Chelex‐100 treated 50 mM MOPS, pH=7.0 to remove the zinc‐EDTA complex. The apoenzyme was reconstituted with 1mM CoSO4 and concentrated by ultrafiltration on an Amicon YM‐10 membrane. Steady‐state kinetic assays were carried out in 50 mM MOPS/1mM CoSO4, pH=7.0; the rate of enzymatic hydrolysis at 30°C was measured using 4.3 mM cephalosporin C as the substrate. The inhibition pattern revealed a non‐competitive mechanism of inhibition for the cobalt form reminiscent of that for the native zinc enzyme. Under the assay conditions described above, the kinetic parameters obtained for the cobalt‐reconstituted form of the B. cereus 5/B/6 metallo‐β‐lactamase were: IC50 = 21μM, Ki = 25 μM and Ki’ = 290 μM.