Catalytic efficiency (kcat/Km)
500 μl of 10 mM HEPES buffer containing 50 μM ZnSO4
The following antibiotics and chemicals were used for the kinetic analysis. Ampicillin (Δε235 = −820 M−1 cm−1) and cefotaxime (Δε260 = −7,500 M−1 cm−1) were purchased from Wako Chemicals (Tokyo, Japan). Benzylpenicillin (Δε235 = −775 M−1 cm−1), cephaloridine (Δε260 = −10,000 M−1 cm−1), ceftazidime (Δε260 = −9,000 M−1 cm−1), imipenem (Δε300 = −9,000 M−1 cm−1), and meropenem (Δε300 = −6,500 M−1 cm−1) were purchased from Sigma-Aldrich (St. Louis, MO). Nitrocefin (Δε482 = 15,000 M−1 cm−1) was purchased from Unipath Oxoid (Basingstoke, United Kingdom). The kinetic analysis was carried out as reported by Borgianni et al. (9). The reaction was performed at 30°C in 500 μl of 10 mM HEPES buffer (pH 7.5) containing 50 μM ZnSO4, and all measurements were made using a Jasco V-530 spectrophotometer. The enzyme was diluted by adding bovine serum albumin (BSA) to the buffer, to a final concentration of 20 μg ml−1, to prevent denaturation. At least three independent progress curves were obtained for each substrate, until reproducible results were obtained.
9. Borgianni L, Prandi S, Salden L, Santella G, Hanson ND, Rossolini GM, Docquier JD. 2011. Genetic context and biochemical characterization of the IMP-18 metallo-β-lactamase identified in a Pseudomonas aeruginosa isolate from the United States. Antimicrob Agents Chemother 55:140–145. doi:10.1128/AAC.00858-10.