title: Continuous fluorescence anisotropy-based assay of BOCILLIN FL penicillin reaction with penicillin binding protein 3.
abstract: We report a simple, rapid, and reproducible fluorescence anisotropy-based method for measuring rate constants for acylation and deacylation of soluble penicillin binding protein (PBP) constructs by compounds in microtiter plates by means of competition with time-dependent acylation by BOCILLIN FL. The method is demonstrated by measuring the acylation rate constants of the PBP3 periplasmic domains from Pseudomonas aeruginosa and Acinetobacter baumannii by BOCILLIN FL, aztreonam, meropenem, and ceftazidime. The new method requires very little protein and can be completed in approximately 1h per compound. A set of BOCILLIN FL acylation progress curves collected over a range of competitor concentrations is fit globally to a kinetic model by numerical integration. First-order deacylation rate constants could also be measured, as demonstrated with a catalytically impaired mutant OXA-10 β-lactamase.
authors.affiliation: Bioscience Department, Infection Innovative Medicines, AstraZeneca R&D Boston, Waltham, MA 02451, USA. adam.shapiro@astrazeneca.com
authors.forename: Adam B
authors.initials: AB
authors.lastname: Shapiro
authors.forename: Rong-Fang
authors.initials: RF
authors.lastname: Gu
authors.forename: Ning
authors.initials: N
authors.lastname: Gao
authors.forename: Stephania
authors.initials: S
authors.lastname: Livchak
authors.forename: Jason
authors.initials: J
authors.lastname: Thresher
mesh_terms: D040981:Acinetobacter baumannii / Q000166:cytology; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D001896:Boron Compounds / Q000378:metabolism*; D005454:Fluorescence Polarization / Q000379:methods*; D007700:Kinetics; D046915:Penicillin-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D010406:Penicillins / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000166:cytology
chemical_list: C118961:BOCILLIN FL; D001426:Bacterial Proteins; D001896:Boron Compounds; D046915:Penicillin-Binding Proteins; D010406:Penicillins