Microcalorimetric studies of rhus vernicifera laccase catalytic oxidation reactions

The oxidation of substrates with various function groups (i.e., ortho-diphenol, ortho-aminophenol, p-aminophenol, ortho-phenylene diamine and p-phenylene diamine) catalyzed by Laccase have been studied using LKB-2107 batch microcalorimetry system. The overall reaction enthalpy ΔrHm, Michaelis constant Km, pseudo-first-order rate constant k2 of the reactions and binding energy ΔG0 of Laccase-substrate complex for each substrate have been determined at 298.15 K, pH 7.4. The binding enthalpy ΔH0 and binding entropy ΔS0 of LS complex, when the substrate is Gallic acid and 2,3-dicyan hydroquinone respectively, have been also determined. The rate constants of the reaction between Laccase and these substrates are essentially identical because the rate of Laccase-catalytic reaction is limited by the step of electron transfer from the type 2 Cu(II)-bound substrate to the less exposed type 1 copper site, and the intra-complex electron transfer rate mainly dependent on the amino residues conformational changes. But the rate of the reaction between Laccase and substrates with group -OH is slower than group -NH2, because of the higher energy of bounding electron. The stability of LS complex depends on ΔS0, since ΔH0>0, substrate distinguished by Laccase depends on the value of allosteric entropy of the amino acid residues induced by substrate. The relationship between Laccase and its substrates cannot be regarded as a simple relationship of lock and key, but an induce-fit relationship.