ISSN:
1432-1327
Keywords:
Key words Galactose oxidase
;
Fusarium
;
Kinetics enzyme reaction
;
Mechanism
;
Variant Tyr495Phe
;
UV-Vis spectroscopy
;
Auto-redox
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
Abstract 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 (k 1), and GOaseredH2+O2→ GOaseox+H2O2 (k 2), 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 k 1 / 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, k 1 values decreased by factors of up to a half, and acid dissociation pK as in the range 6.6–6.9 were obtained. UV-Vis spectrophotometric studies on GOaseox gave an independently determined pK a 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 pK a is therefore assigned to protonation of Tyr-495 which is a ligand to the Cu. The rate constant k 2 (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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/s007750050159
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