Steady-state and transient-state kinetic studies on the oxidation of 3,4-dimethoxybenzyl alcohol catalyzed by the ligninase of Phanerochaete chrysosporium burds

Catalysis of the H₂-O₂-dependent oxidation of 3,4-dimethoxybenzyl (veratryl) alcohol by the hemoprotein ligninase isolated from wood-decaying fungus, Phanerochaete chrysosporium Burds, is characterized. The reaction yields veratraldehyde and exhibits a stoichiometry of one H₂O₂ consumed per aldehyde formed. Ping-pong steady-state kinetics are observed for H₂O₂ (K(M) = 29 μM) and veratryl alcohol (K(M) = 72 μM) at pH 3.5. The magnitude of the turnover number varied from 2 to 3 s^-1 at this pH, depending on the preparation of the enzyme. Each preparation of enzyme consists of a mixture of active and inactive enzyme. Extensive steady-state kinetic studies of several different preparations of enzyme, suggest a mechanism in which H₂O₂ reacts with enzyme to form an intermediate that subsequently reacts with the alcohol to return the enzyme to the resting state. The pH dependence of the overall reaction indicates that an ionization occurs having an apparent pK(a) ~3.1. The activity is, thus, nearly zero at pH 5 and increases to a maximum near pH ~2. However, the enzyme is unstable at this low pH. Transient-state kinetic studies reveal that, upon reaction of ligninase with H₂O₂, spectral changes occur in the Soret region, which, by analogy to previous studies of horseradish peroxidase, are consistent with formation of Compounds I and II. The active form of the enzyme appears to react rapidly with H₂O₂; we observed a positive correlation between the turnover number of the enzyme preparation and the extent of a rapid reaction between H₂O₂ and ligninase to form Compound I. Free radical cations derived from veratryl alcohol do not appear to be released from the enzyme during catalysis; however, other substrates are known to be converted to cation radicals (Kersten, P., Tien, M., Kalyanaraman, B., and Kirk, T.K. (1985) J. Biol. Chem. 260, 2609-2612). Our results are generally consistent with a classical peroxidase mechanism for the action of ligninase on lignin-like substrates.