Pre-steady-state kinetic study on the formation of Compound I and II of ligninase |
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| Institution: | 1. Imperial College of Science & Technology, Pure and Applied Biology, Prince Consort Road, London, U.K.;2. Laboratory of Biochemistry and Biotechnological centre, University of Amsterdam, Amsterdam, The Netherlands;1. Electrochemical Methods Laboratory - Analytical and Applied Chemistry Department at Institut Químic de Sarrià, Universitat Ramon Llull, Via Augusta, 390, 08017, Barcelona, Spain;2. Grup d’Enginyeria de Materials (GEMAT) at Institut Químic de Sarrià, Universitat Ramon Llull, Via Augusta, 390, 08017, Barcelona, Spain;3. CIBER-BBN, Networking Center on Bioengineering, Biomaterials and Nanomedicine, Zaragoza, Spain;1. Department of Chemistry, Faculty of Science, Yazd University, Yazd, 89195-741, Iran;2. Department of Biology, Faculty of Science, Yazd University, Yazd, 89195-741, Iran;1. Aalto University School of Chemical Technology, Department of Forest Products Technology, P.O. Box 16300, Aalto, 00076, Finland;2. Leibniz-Institute of Agricultural Engineering Potsdam-Bornim, Department of Post Harvesting Technologies, Germany;3. Hochschule Bremen-University of Applied Sciences, Faculty 5, Biomimetics—The Biological Materials Group, Neustadtswall 30, 28199 Bremen, Germany;1. School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA, USA;2. Department of Chemical and Biological Engineering, Korea University, Seoul 02841, Republic of Korea |
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| Abstract: | The reaction between ligninase and hydrogen peroxide yielding Compound I has been investigated using a stopped-flow rapid-scan spectrophotometer. The optical absorption spectrum of Compound I appears different to that reported by Andrawis, A. et al. (1987) and Renganathan, V. and Gold, M.H. (1986), in that the Soret-maximum is at 401 nm rather than 408 nm. The second-order rate constant (4.2·105 M−1·s−1) for the formation of Compound I was independent of pH (pH 3.0–6.0). In the absence of external electron donors, Compound I decayed to Compound II with a half-life of 5–10 s at pH 3.1. The rate of this reaction was not affected by the H2O2 concentration used. In the presence of either veratryl alcohol or ferrocyanide, Compound II was rapidly generated. With ferrocyanide, the second-order rate constant increased from 1.9·104 M−1·s−1 to 6.8·106 M−1·s−1 when the pH was lowered from 6.0 to 3.1. With veratryl alcohol as an electron donor, the second-order rate constant for the formation of Compound II increased from 7.0·103 M−1·s−1 at pH 6.0 to 1.0·105 M−1·s−1 at pH 4.5. At lower pH values the rate of Compound II formation no longer followed an exponential relationship and the steady-state spectral properties differed to those recorded in the presence of ferrocyanide. Our data support a model of enzyme catalysis in which veratryl alcohol is oxidized in one-electron steps and strengthen the view that veratryl alcohol oxidation involves a substrate-modified Compound II intermediate which is rapidly reduced to the native enzyme. |
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