Reactions of lignin peroxidase compounds I and II with veratryl alcohol: Transient-state kinetic characterization

Hiroyuki Wariishi, Jin Huang, H. Brian Dunford, Michael H. Gold

Research output: Contribution to journalArticle

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Abstract

Stopped-flow techniques were utilized to investigate the kinetics of the reaction of lignin peroxidase compounds I and II (LiPI and LiPII) with veratryl alcohol (VA). All rate data were collected from single turnover experiments under pseudo first-order conditions. The reaction of LiPI with VA strictly obeys second-order kinetics over the pH range 2.72-5.25 as demonstrated by linear plots of the pseudo first-order rate constants versus concentrations of VA. The second-order rate constants are strongly dependent on pH and range from 2.62 × 106 M-1 s-1 (pH 2.72) to 1.45 × 104 M-1 s-1 (pH 5.25). The reaction of LiPII and VA exhibits saturation behavior when the observed pseudo first-order rate constants are plotted against VA concentrations. The saturation phenomenon is quantitatively explained by the formation of a 1:1 LiPII-substrate complex. Results of kinetic and rapid scan spectral analyses exclude the formation of a LiPII-VA cation radical complex. The first-order dissociation rate constant and the equilibrium dissociation constant for the LiPII reaction are also pH dependent. Binding of VA to LiPII is controlled by a heme-linked ionizable group of pKa ∼ 4.2. The pH profiles of the second-order rate constants for the LiPI reaction and of the first-order dissociation constants for the LiPII reaction both demonstrate two pKa values at ∼3.0 and ∼4.2. Protonated oxidized enzyme intermediates are most active, suggesting that only electron transfer, not proton uptake from the reducing substrate, occurs at the enzyme active site. These results are consistent with the one-electron oxidation of VA to an aryl cation radical by LiPI and LiPII.

Original languageEnglish
Pages (from-to)20694-20699
Number of pages6
JournalJournal of Biological Chemistry
Volume266
Issue number31
Publication statusPublished - Nov 5 1991
Externally publishedYes

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Kinetics
Rate constants
Cations
Electrons
Proton transfer
veratryl alcohol
lignin peroxidase
Substrates
Enzymes
Heme
Protons
Catalytic Domain
Oxidation
Experiments

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Reactions of lignin peroxidase compounds I and II with veratryl alcohol : Transient-state kinetic characterization. / Wariishi, Hiroyuki; Huang, Jin; Dunford, H. Brian; Gold, Michael H.

In: Journal of Biological Chemistry, Vol. 266, No. 31, 05.11.1991, p. 20694-20699.

Research output: Contribution to journalArticle

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