Entropy drives selective fluorine recognition in the fluoroacetyl-CoA thioesterase from <i>Streptomyces cattleya</i>.

Abstract

Fluorinated small molecules play an important role in the design of bioactive compounds for a broad range of applications. As such, there is strong interest in developing a deeper understanding of how fluorine affects the interaction of these ligands with their targets. Given the small number of fluorinated metabolites identified to date, insights into fluorine recognition have been provided almost entirely by synthetic systems. The fluoroacetyl-CoA thioesterase (FlK) from <i>Streptomyces cattleya</i> thus provides a unique opportunity to study an enzyme-ligand pair that has been evolutionarily optimized for a surprisingly high 10⁶ selectivity for a single fluorine substituent. In these studies, we synthesize a series of analogs of fluoroacetyl-CoA and acetyl-CoA to generate nonhydrolyzable ester, amide, and ketone congeners of the thioester substrate to isolate the role of fluorine molecular recognition in FlK selectivity. Using a combination of thermodynamic, kinetic, and protein NMR experiments, we show that fluorine recognition is entropically driven by the interaction of the fluorine substituent with a key residue, Phe-36, on the lid structure that covers the active site, resulting in an ∼5- to 20-fold difference in binding (<i>K</i>_D). Although the magnitude of discrimination is similar to that found in designed synthetic ligand-protein complexes where dipolar interactions control fluorine recognition, these studies show that hydrophobic and solvation effects serve as the major determinant of naturally evolved fluorine selectivity.