3: Chemical tools to determine the mechanism of cargo-flipping in ACP-dependent enzymes

Abstract: Acyl Carrier Proteins (ACPs) are essential components of fatty acid biosynthetic and quorum-sensing signaling pathways. A key mechanistic feature of ACPs is chain flipping, in which the acyl cargo transitions from a sequestered state within the carrier protein core to an exposed conformation in the enzyme active site. Despite its importance in substrate recognition, quantitative characterization of chain-flipping kinetics remains limited. This study focuses on investigating the chain flipping mechanism of ACPs using Alkyl ACP and S-adenosyl-L-methionine (SAM) as substrates for the Acyl Homoserine Lactone (AHL) Synthase enzyme RhlI. Short and medium length chain alkyl-ACP substrates were synthesized to probe substrate-dependent dynamics. The interaction between ACP and RhlI is expected to modulate cargo accessibility, providing insights into enzyme specificity and catalytic efficiency. Cargo translocation was directly observed using stopped-flow fluorescence spectroscopy. The kon and koff of acyl-chain cargo flipping on to the partner enzyme should offer direct kinetic insights into ACP-mediated substrate delivery and establish a quantitative framework for understanding the interactions between carrier proteins and enzymes in critical biological pathways.