Molecular Mechanisms Underlying Inhibitory Binding of Alkylimidazolium Ionic Liquids to Laccase.

Water-miscible alkylimidazolium ionic liquids (ILs) are "green" co-solvents for laccase catalysis, but generally inhibit enzyme activity. Here, we present novel insights into inhibition mechanisms by a combination of enzyme kinetics analysis and molecular simulation. Alkylimidazolium cations competitively bound to the TI Cu active pocket in the laccase through hydrophobic interactions. Cations with shorter alkyl chains (C₂~C₆) entered the channel inside the pocket, exhibiting a high compatibility with laccase (competitive inhibition constant Kic = 3.36~3.83 mM). Under the same conditions, [Omim]Cl (Kic = 2.15 mM) and [Dmim]Cl (Kic = 0.18 mM) with longer alkyl chains bound with Leu296 or Leu297 near the pocket edge and Leu429 around TI Cu, which resulted in stronger inhibition. Complexation with alkylimidazolium cations shifted the pH optima of laccase to the right by 0.5 unit, and might, thereby, lead to invalidation of the Hofmeister series of anions. EtSO₄- showed higher biocompatibility than did Ac- or Cl-, probably due to its binding near the TI Cu and its hindering the entry of alkylimidazolium cations. In addition, all tested ILs accelerated the scavenging of 2, 2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radicals, which, however, did not play a determining role in the inhibition of laccase.