Biochemical analysis of relebactam inhibition of M. abscessus \(\beta \)-lactamase, BlaMab. (2a) Our novel Thin Layer Chromatography (TLC) assay exhibiting the activity of BlaMab in the turnover of penicillin V (high Rf value) to penicilloic acid (lower Rf value). In the absence, or termination of activity of BlaMab (by boiling (100 °C) for 1 h) or addition of known inhibitor avibactam19,27 (200 µg/mL) no lower Rf value spot corresponding to penicilloic acid is resolved by TLC. The addition of relebactam to the reaction between BlaMab and penicillin V also results in the absence of the lower Rf value spot. (2b) This observed inhibition is validated by a spectrophotometric analysis. The increase in concentration of relebactam resulted in partial inhibition of nitrocefin turnover at 1 µM and total abrogation at 10 µM. The initial velocity (vi) of the reaction between BlaMab and nitrocefin was monitored for a range of substrate concentrations (1–500 µM) and relebactam concentrations (0, 0.5, 0.75, 1 and 2.5 µM). (2c) This data was plotted vi vs [S] in order to determine Km values. (2d) The values for kobs were obtained as previously described19 and plotted against relebactam concentrations ([I]) to deduce a carbamylation rate (k2/Ki) for BlaMab. (2e) The kinetics of BlaMab decarbamylation were assessed to show the recovery of nitrocefin hydrolysis by BlaMab after inhibition by relebactam in order to derive a koff value. (2 f) Kinetic parameters were derived as described previously19.