A role of Gln596 in fine-tuning mammalian ALOX15 specificity, protein stability and allosteric properties

His596 of human ALOX12 has been suggested to interact with the COO−-group of arachidonic acid during ALOXcatalysis. In mammalian ALOX15 orthologs Gln596 occupies this position and this amino acid exchange mightcontribute to the functional differences between the two ALOX-isoforms. To explore the role of Gln596 forALOX15 functionality we mutated this amino acid to different residues in rabbit and human ALOX15 and investigatedthe impact of these mutations on structural, catalytic and allosteric enzyme properties. To shed lighton the molecular basis of the observed functional alterations we performed in silico substrate docking studies andmolecular dynamics simulations and also explored the impact of Gln596 exchange on the protein structure. Thecombined theoretical and experimental data suggest that Gln596 may not directly interact with the COO−-groupof arachidonic acid. In contrast, mutations at Gln596 destabilize the secondary and tertiary structure of ALOX15orthologs, which may be related to a disturbance of the electrostatic interaction network with other amino acidsin the immediate surrounding. Moreover, our MD-simulations suggest that the geometry of the dimer interfacedepends on the structure of substrate bound inside the substrate-binding pocket and that Gln596Ala exchangeimpairs the allosteric properties of the enzyme. Taken together, these data indicate the structural and functionalimportance of Gln596 for ALOX15 catalysis.