KINETIC-STUDIES ON THE REACTIONS OF FUSARIUM GALACTOSE-OXIDASE WITH 5DIFFERENT 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 R CH2OH:- 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 pre sence 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 re actions, monitored at 450 nm by stopped-flow spectrophotometry, termin ated 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 GO ase(red)H(2) + O-2 --> GOase(ox) + H2O2 (k(2)), with GOase(red)H, writ ten 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) ''nat ive'' 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 decrease d by factors of up to a half, and acid dissociation pK(a)s in the rang e 6.6-6.9 were obtained. UV-Vis spectrophotometric studies on GOase,, gave an independently determined pK(a) of 6.7. No corresponding reacti ons of the Tyr495Phe variant were observed, and there are no similar U V-Vis absorbance changes for this variant. The pK(a) is therefore assi gned to protonation of Tyr-495 which is a ligand to the Cu. The rate c onstant k(2) (1.01 x 10(7) M(-1)s(-1)) is independent of pH in the ran ge 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 num ber of three is likely.