Km

Activity

Michaelis Constant (Km)

microM

Spectrophotometric/Colorimetric Assay

50 mM HEPES buffer (pH 7.2) supplemented with 1 μg/mL BSA, 1 μM ZnSO4 and 0.01% Triton X-100

25°C

7.2

Variable

Variable

None

The hydrolysis of various β-lactam substrates was monitored at 25°C in 50 mM HEPES buffer (pH 7.2) supplemented with 1 μg/mL BSA, 1 μM ZnSO4 and 0.01% Triton X-100. (35) For ampicillin hydrolysis, 50 mM MOPS buffer (pH 7.2) was used due to the high background hydrolysis of this penicillin in HEPES buffer. (36) Analyses were carried out in triplicate (n ≥ 3); the absorbance values were read using a BMG Labtech Pherastar FS plate reader. Extinction coefficients were determined by plotting the absorbance units against increasing concentrations of the substrates or product. Kinetic constants (KM and kcat) were obtained by determining the initial rate of the reaction at different substrate concentrations. The concentration-dependence of the initial rate was fitted and analysed using GraphPad Prism® 5.01 software to generate Michaelis–Menten and substrate inhibition curves.

Reference:
35 van Berkel SS Brem J Rydzik AM et al. Assay platform for clinically relevant metallo-β-lactamases J Med Chem 2013 56 6945 53

36 Horton LB Shanker S Mikulski R et al. Mutagenesis of zinc ligand residue Cys221 reveals plasticity in the IMP-1 metallo-β-lactamase active site Antimicrob Agents Chemother 2012 56 5667 77