How Can Linoleic Acid Be the Preferential Substrate of the Enzyme 15-Lipoxygenase-1? A QM/MM Approach

The most common substrate of mammalian lipoxygenases (LOXs) is arachidonic acid (AA). However, 15-LOXs can present dual substrate specificity. These LOXs catalyze the peroxidation of AA, initiated by a H-abstraction step (mainly H₁₃-abstraction) by the Fe(III)-OH^- cofactor, and the peroxidation of linoleic acid (LA) after H₁₁-abstraction. In this paper, QM(B3LYP)/MM(CHARMM) calculations of the rate-limiting H₁₁-abstraction process of LA catalyzed by rabbit 15-LOX-1 (15-rLOX-1) have been carried out using a complete model of the solvated 15-rLOX-1:LA complex. A total of 26 QM/MM potential energy profiles as a function of the H-transfer reaction coordinate have been computed along with one QM/MM free energy profile obtained using the Free Energy Perturbation method. The molecular origin of substrate specificity of 15-rLOX-1 for LA in comparison with AA has been analyzed. In many of the QM/MM reactive 15-rLOX-1:LA energy minima, LA adopts more elongated conformations than AA, although having a shorter carbon chain, because LA has one double bond between C₁ and C₁₁ whereas AA has three double bonds between C₁ and C₁₃. Consequently, C₁₁ of LA can be located in the same region of the active site as C₁₃ of AA, a zone where H₁₁-abstraction from LA as well as H₁₃-abstraction from AA is not hindered by bulky residue side chains. This explains at a molecular level how 15-LOXs might accommodate and recognize for catalysis two substrates that are different in length by two carbons. Our results also explain why (9Z,11E)-13-hydro(pero)xyoctadeca-9,11-dienoic acid is the major product of the peroxidation and why LA is the preferential substrate of 15-rLOX-1.