Previous comparative kinetic isotope effects have inferred an allosteric site for fatty acids and their derivatives that modulates substrate selectivity in 15lipoxygenases. Hydrogen-deuterium exchange also previously revealed regionally defined enhanced protein flexibility, centred at helix a2a 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 a2.