kcat

Activity

Catalytic Rate Constant (kcat)

1/s

Spectrophotometric/Colorimetric Assay

500 μl of 10 mM HEPES buffer containing 50 μM ZnSO4

30°C

7.5

Variable

Variable

None

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.

Reference:
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.