LAUSR

Previous comparative kinetic isotope effects have inferred an allosteric site for fatty acids and their derivatives that modulates substrate selectivity in 15‐lipoxygenases. Hydrogen–deuterium exchange also previously revealed regionally defined enhanced protein flexibility, centred at helix α2 – a gate to the substrate entrance. Direct evidence for allosteric binding and a complete understanding of its mechanism remains elusive. In this study, we examine the binding thermodynamics of the fatty acid mimic, oleyl sulfate (OS), with the monomeric model plant 15‐LOX, soybean lipoxygenase (SLO), using isothermal titration calorimetry. Dynamic light scattering and differential scanning calorimetry rule out OS‐induced oligomerization or structural changes. These data provide evidence that the fatty acid allosteric regulation of SLO is controlled by the dynamics of helix α2.