<i>k</i><sub>inact</sub>/<i>K</i><sub>I</sub> Value Determination\nfor Penicillin-Binding Proteins\nin Live Cells

Penicillin-binding proteins (PBPs) are an essential family\nof bacterial\nenzymes that are covalently inhibited by the β-lactam class\nof antibiotics. PBP inhibition disrupts peptidoglycan biosynthesis,\nwhich results in deficient growth and proliferation, and ultimately\nleads to lysis. IC<sub>50</sub> values are often employed as descriptors\nof enzyme inhibition and inhibitor selectivity, but can be misleading\nin the study of time-dependent, covalent inhibitors. Due to this disconnect,\nthe second-order rate constant, <i>k</i><sub>inact</sub>/<i>K</i><sub>I</sub>, is a more appropriate metric of\ncovalent-inhibitor potency. Despite being the gold standard measurement\nof potency, <i>k</i><sub>inact</sub>/<i>K</i><sub>I</sub> values are typically obtained from <i>in vitro</i> assays, which limits assay throughput if investigating an enzyme\nfamily with multiple homologues (such as the PBPs). Therefore, we\ndeveloped a whole-cell <i>k</i><sub>inact</sub>/<i>K</i><sub>I</sub> assay to define inhibitor potency for the\nPBPs in <i>Streptococcus pneumoniae</i> using the fluorescent,\nactivity-based probe, Bocillin-FL. Our results align with <i>in vitro k</i><sub>inact</sub>/<i>K</i><sub>I</sub> data and show a comparable relationship to previously established\nIC<sub>50</sub> values. These results support the validity of our <i>in vivo k</i><sub>inact</sub>/<i>K</i><sub>I</sub> method as a means of obtaining β-lactam potency for a suite\nof PBPs to enable structure–activity relationship studies.