/ Value Determination for Penicillin-Binding Proteins in Live Cells.

Penicillin-binding proteins (PBPs) are an essential family of bacterial enzymes that are inhibited by the β-lactam class of antibiotics. PBP inhibition disrupts cell wall biosynthesis, which results in deficient growth and proliferation, and ultimately leads to lysis. IC values are often employed as descriptors of enzyme inhibition and inhibitor selectivity but can be misleading in the study of time-dependent, irreversible inhibitors. Due to this disconnect, the second order rate constant / is a more appropriate metric of covalent inhibitor potency. Despite being the gold standard measurement of potency, / values are typically obtained from assays, which limits assay throughput if investigating an enzyme family with multiple homologs (such as the PBPs). Therefore, we developed a whole-cell / assay to define inhibitor potency for the PBPs in using the fluorescent activity-based probe Bocillin-FL. Our results align with / data and show a comparable relationship to previously established IC values. These results support the validity of our / method as a means of obtaining a full picture of β-lactam potency for a suite of PBPs.