Kinetic Analysis of TEM β-Lactamase Variants

The wt and mutant TEM variants were generated as outlined in the ESI.† The proteins were expressed in E. coli using a bespoke plasmid based on pBAD called pBADKAN, and purified as described in the ESI.† The TEM-dependent kinetics of ampicillin hydrolysis (ε₂₃₅ = 1500 M^–1 cm^–1) were determined spectrophotometrically using 1 cm path length QS quartz cuvette (Hellma). Hydrolysis assays were carried out in a 1 mL reaction volume. Purified enzyme was diluted to a final concentration of 250 ng μL^–1 in 50 mM sodium phosphate buffer, pH 8 at room temperature. Reactions were started by addition of ampicillin and hydrolysis was measured by the decrease in absorbance at 235 nm. Ampicillin concentrations ranged from 50 μM to 800 μM. Kinetic parameters were calculated using initial rate of hydrolysis at each substrate concentration and then fitting to the Michaelis–Menten equation using GraphPad Prism. TEM β-lactamase activity using the colorimetric substrate nitrocefin was performed essentially as described previously.^{47 (link),48 (link)}