Kinetic studies on the reactions of Fusarium galactose oxidase with five different substrates in the presence of dioxygen

Autor: Christopher D. Borman, A. G. Sykes, Colin G. Saysell
Rok vydání: 1997
Předmět:
Zdroj: JBIC Journal of Biological Inorganic Chemistry. 2:480-487
ISSN: 1432-1327
0949-8257
DOI: 10.1007/s007750050159
Popis: Reactions (25 °C) of galactose oxidase, GOaseox from Fusarium NRRL 2903 with five different primary-alcohol-containing substrates RCH2OH:- D-galactose (I) and 2-deoxy-d-galactose (II) (monosaccharides); methyl-β-d-galactopyranoside (III) (glycoside);d-raffinose (IV) (trisaccharide); and dihydroxyacetone (V) have been studied in the presence of O2. The GOaseox state has a tyrosyl radical coordinated at a square-pyramidal CuII active site, and is a two-equivalent oxidant. Reactant concentrations were [GOaseox] (0.8–10 μM), RCH2OH (1.0–6.0 mM), and O2 (0.14–0.29 mM), with I=0.100 M (NaCl). The reactions, monitored at 450 nm by stopped-flow spectrophotometry, terminated with depletion of the O2. Each trace was fitted to the competing reactions GOaseox+RCH2 OH → GOaseredH2+RCHO (k1), and GOaseredH2+O2→ GOaseox+H2O2 (k2), with GOaseredH2 written as the doubly protonated two-electron-reduced CuI product. It was necessary to avoid auto-redox interconversion of GOaseox and GOasesemi . Information obtained at pH 7.5 indicates a 5 : 95 (ox : semi) "native" mix equilibration complete in ∼3 h. At pH >7.5, rate constants 10–4 k1 / M–1 s–1 for the reactions of GOaseox with (I) (1.19), (II) (1.07), (III) (1.29), (IV) (1.81), (V) (2.94) were determined. On decreasing the pH to 5.5, k1 values decreased by factors of up to a half, and acid dissociation pKas in the range 6.6–6.9 were obtained. UV-Vis spectrophotometric studies on GOaseox gave an independently determined pKa of 6.7. No corresponding reactions of the Tyr495Phe variant were observed, and there are no similar UV-Vis absorbance changes for this variant. The pKa is therefore assigned to protonation of Tyr-495 which is a ligand to the Cu. The rate constant k2 (1.01×107 M–1 s–1) is independent of pH in the range 5.5–9.0 investigated, suggesting that H+ (or H-atoms) for the O2 → H2O2 change are provided by the active site of GOasered . The CuI of GOasered is less extensively complexed, and a coordination number of three is likely.
Databáze: OpenAIRE
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