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

Penicillin-binding proteins (PBPs) are an essential family of bacterial enzymes that are covalently inhibited by the β-lactam class of antibiotics. PBP inhibition disrupts peptidoglycan biosynthesis, which results in deficient growth and proliferation, and ultimately leads to lysis. IC₅₀ values are often employed as descriptors of enzyme inhibition and inhibitor selectivity, but can be misleading in the study of time-dependent, covalent inhibitors. Due to this disconnect, the second-order rate constant, k_inact/K_I, is a more appropriate metric of covalent-inhibitor potency. Despite being the gold standard measurement of potency, k_inact/K_I values are typically obtained from in vitro assays, which limits assay throughput if investigating an enzyme family with multiple homologues (such as the PBPs). Therefore, we developed a whole-cell k_inact/K_I assay to define inhibitor potency for the PBPs in Streptococcus pneumoniae using the fluorescent, activity-based probe, Bocillin-FL. Our results align with in vitro k_inact/K_I data and show a comparable relationship to previously established IC₅₀ values. These results support the validity of our in vivo k_inact/K_I method as a means of obtaining β-lactam potency for a suite of PBPs to enable structure-activity relationship studies.