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

Jianliang Sun,Shiyu Fu,Shicheng Chen,Hao Liu,Wenping Yang

doi:10.3390/molecules22081353

Abstract

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.

Highlights

Laccase (EC 1.10.3.2) is an efficient biocatalyst for synthesizing dimers, polymers and composites from a diverse range of substrates including phenolics, aromatic amines, flavonoids, acrylamide and thiols [1,2]
Our results indicated that a competitive inhibition mechanism existed in the interactions between Myceliophthora thermophila (Mth) laccase and alkylimidazolium chlorides (Figure 2)
The alkylimidazolium cations competitively bound to the TI Cu active pocket of Mth laccase

Summary

Introduction

Laccase (EC 1.10.3.2) is an efficient biocatalyst for synthesizing dimers, polymers and composites from a diverse range of substrates including phenolics, aromatic amines, flavonoids, acrylamide and thiols [1,2]. Laccase-induced synthesis is often conducted in aqueous mixtures of organic solvents, e.g., acetone and methanol, to promote the yield and/or molecular weight of products [3,4]. Many laccases are tolerant to these solvents at a high content, e.g., 50% (v/v) [4,5]. The use of conventional volatile solvents threatens the environment [6]. Selected ILs could further lessen the inactivation of laccase by the template molecules during aniline polymerization [8], or by the oxidative form of redox mediators [9]. Most water-miscible ILs for potential use in homogeneous synthesis have been identified as laccase inhibitors (Figure 1) except several particular species, e.g., [Emim]EtSO4 and [TMA]TfO, under specified conditions [8,10,11,12]

Methods

Results

Discussion

Conclusion