Abstract
The steady state kinetic parameters Km and kcat for the oxidation of phenolic substrates by lignin peroxidase correlated with the presteady state kinetic parameters Kd and k for the reaction of the enzyme intermediate compound II with the substrates, indicating that the latter is the rate-limiting step in the catalytic cycle. ln Km and ln Kd values for phenolic substrates correlated with redox properties, unlike ln kcat and ln k. This finding suggests that in contrast to horseradish peroxidase, electron transfer is not the rate-limiting step during oxidation by lignin peroxidase compound II. A mechanism is proposed for lignin peroxidase compound II reactions consisting of an equilibrium electron transfer step followed by a subsequent rate-limiting step. Analysis of the correlation coefficients for linear relationships between ln Kd and ln Km and different calculated redox parameters supports a mechanism in which the acidic forms of phenols are oxidized by lignin peroxidase and electron transfer is coupled with proton transfer. 1,2-Dimethoxyarenes did not comply with the trend for phenolic substrates, which may be a result of more than one substrate binding site on lignin peroxidase and/or alternative binding modes. This behavior was supported by analogue studies with the 1,2-dimethoxyarenes veratric acid and veratryl aldehyde, both of which are not oxidized by lignin peroxidase. Inclusion of either had little effect on the rate of oxidation of phenolic substrates yet resulted in a decrease in the oxidation rate of 1,2-dimethoxyarene substrates, which was considerable for veratryl alcohol and less pronounced for 3,4-dimethoxyphenethylalcohol and 3,4-dimethoxycinnamic acid, in particular in the presence of veratric acid.
Highlights
Lignin peroxidases (LIP)1 play a central role in the biodegradation of the plant cell wall constituent lignin by white-rot
Kinetic studies indicate a hyperbolic concentration dependence for the conversion of LIP-compound II (LIPII) to Fe-LIP by guaiacol [4, 5], -O-4-Phenolic lignin oligomers [5], ferulic acid [6], and nonphenolic veratryl alcohol [17], which suggests at least a two-step mechanism for this stage
Rapid equilibrium is ascribed to binding of a substrate molecule to LIPII, and the rate-limiting step is commonly considered as an electron transfer from the bound substrate molecule to LIPII [4, 5]
Summary
Enzyme Purification—LIP isoenzyme H1 was produced from high nitrogen cultures of P. chrysosporium Burds BKM-F-1767 [27] and purified as described previously [6]. The amount of substrate consumed in 10 s (calculated from blanks without H2O2 and authentic compounds) for different initial substrate concentrations was determined. Three replicates of both blanks and reactions were analyzed. The amount of substrate consumed in 10 s was calculated from blanks without H2O2 and authentic compounds The UHF and ROHF adiabatic ionization potentials (IPad(UHF) and IPad(ROHF), respectively) were calculated as the difference between the heats of formation of the substrates and their radical cations after the optimal geometries were determined. The extinction coefficients were determined by preparing a series of solutions of increasing concentrations for each substrate and measuring the absorbance at the predetermined wavelength of maximum absorbance (max). All stock solutions were 100 mM except 3,4-dimethoxycinnamic acid, which was 10 mM
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