Substrate stabilization and noncompetitive inhibition effects of a water-miscible ionic liquid [BMPy][BF4] in the catalysis of horseradish peroxidase

Abstract

The inhibition mechanism of a water-miscible ionic liquid, N-butyl-3-methypyridinium tetrafluoroborate ([BMPy][BF4]), on the catalysis of horseradish peroxidase (HRP) was investigated. The Km value for the oxidation of guaiacol (2-methoxyphenol) with H2O2 catalyzed by HRP increased from 2.8 mM in 100% water to 12.6 mM in 25% (v/v) [BMPy][BF4]. This increase of Km by the ionic liquid was elucidated to be caused by the strong stabilization of the ground state of guaiacol by the ionic liquid. On the contrary, the kcat value for the HRP-catalyzed reaction decreased from 13.8/sec in 100% water to 6.7/sec in 25% (v/v) [BMPy][BF4]. Such decrease of kcat value of HRP catalysis by the increasing content of [BMPy][BF4] was described using the noncompetitive inhibition of the enzyme by the ionic liquid. The value of the inhibition constant of [BMPy][BF4] was 1.48 M indicating that the ionic liquid exerts a weak noncompetitive inhibition effect on the HRP catalysis.