Abstract Gram-negative bacteria use acyl-homoserine lactone mediated quorum sensing to regulate key physiological activities that includes virulence, biofilm formation and toxin production. AHL synthases make AHL autoinducer signals by enzymatically coupling acyl-ACP and S-adenosyl-L-methionine metabolites to facilitate quorum sensing for the bacterium. Therefore, AHL synthase inhibitors hold significant promise as antimicrobials in treating multidrug resistant bacterial infections. Designing AHL synthase specific inhibitors, however, does remain a significant challenge. To develop QS selective inhibitors, we investigate unique functional aspects of AHL synthases that distinguish them from other enzymes utilizing either acyl-ACP or SAM substrates. One such unique aspect of the synthase is it’s ability to selectively bind and react with a specific acyl-ACP substrate thereby enforcing fidelity in AHL signal synthesis. In general, acyl-ACP binding to a partner enzyme is a minimal two-step process involving an initial electrostatic docking of ACP acidic residues on to a basic patch in the synthase (protein-protein binding) followed by the translocation of the acyl- chain cargo from the ACP to the enzyme’s active site pocket through a process referred to as “chain-flipping”. Decoupling the binding from the chain-flipping step was not possible until now due to lack of methods that could independently report on the chain flipping step in ACP utilizing enzyme reactions. To address this limitation, we placed a site-specific fluorescent label on the EsaI AHL synthase, conducted enzyme-ACP titrations in the pre-steady state kinetics regime using a stopped flow fluorometer and determined kon and koff for both fluorescent and non-fluorescent cargo chain flipping from ACP to the EsaI synthase. Building upon our preliminary data, we propose two aims in this application to validate a fluorescence based method for determining chain-flipping rate constants in AHL synthase enzymes. Successful completion of the proposed studies should a) enhance our understanding on the mechanistic basis of substrate recognition in AHL synthases b) aid in development of a fluorescence-based HTS assay for screening AHL synthase inhibitors and c) open doors for single molecule enzymological investigations on chain-flipping step in AHL synthesis and d) facilitate deployment of this tool to investigate chain-flipping kinetics over a broader range of medicinally important, carrier protein dependent enzymes.