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

Reactions (25 degrees C) of galactose oxidase, GOase(ox) from Fusarium NRRL 2903 with five different primary-alcohol-containing substrates RCH2OH:- D-galactose (I) and 2-deoxy-D-galactose (II) (monosaccharides); methyl-beta-D-galactopyranoside (III) (glycoside); D-raffinose (IV) (trisaccharide); and dihydroxyacetone (V) have been studied in the presence of O-2. The GOase(ox) state has a tyrosyl radical coordinated at a square-pyramidal Cu-II active site, and is a two-equivalent oxidant. Reactant concentrations were [GOase(ox)] (0.8 - 10 mu M), RCH2OH (1.0 - 6.0 mM), and O-2 (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 O-2 Each trace was fitted to the competing reactions GOase(ox) + RCH2 OH --> GOase(red)H(2) + RCHO (k(1)), and GOase(red)H(2) + O-2 --> GOase(ox) + H2O2 (k(2)), with GOase(red)H, written as the doubly protonated two-electron-reduced Cu-I product. It was necessary to avoid auto-redox interconversion of GOase(ox) and GOase(semi). Information obtained at pH 7.5 indicates a 5.95 (ox:semi) ''native'' mix equil ibration complete in similar to 3 h. At pH > 7.5, rate constants 10(-4)k(1)/M(-1)s(-1) for the reactions of GOase(ox) with (I) were determined. On decreasing the pH to 5.5, k(1) val ues decreased by factors of up to a half, and acid dissociation pK(a)s in the range 6.6-6.9 were obtained. UV-Vis spectrophotometric studies on GOase,, 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 x 10(7) M(-1)s(-1)) is independent of pH in the range 5.5 - 9.0 investigated, suggesting that H+ (or H-atoms) for the O-2 --> H2O2 change are provided by the active site of GOase(red). The Cu-I of GOase(red) is less extensively complexed, and a coordination number of three is likely.