Reactivity of galactose oxidase

Recent studies on reactions of the two-equivalent Cu-II/tyrosyl radical containing enzyme galactose oxidase (GOase) from Fusarium NRRL 2903 are referred to in this report. Two GOase(ox) active-site acid dissociation pK(a) values have been determined by UV-vis spectrophotometry, and are 5.7 (coordinated H2O) and 7.0 (protonated Tyr-495). The active enzyme (GOase(ox)) catalyses the oxidation of the primary alcohols RCH2OH + O-2 --> RCHO + H2O2, where previous studies with five different substrates are extended to include saturation kinetics for D-Galactose and D-Raffinose. Two competing steps, GOase(ox) + RCH2OH --> GOase(red)H(2) + RCHO (k(1)) and GOase(red)H(2) + O-2 --> GOase(ox) + H2O2 (k(2)) are observed. Rate constants k(1) are dependent on pH, whereas Ic, is independent of pH in the range 5.5-8.0. The Ic, behaviour suggests that the two protons required to bring about the O-2 --> H2O2 conversion are provided by the protonated form GOase(red)H(2). Michaelis-Menten kinetics allow K-m ( = 1/K-bind) and k(cat) (catalytic turnover) to be determined, where K-bind M-1 values are small for D-Galactose (6.7) and D-Raffinose (14.3), in keeping with the enzyme being extracellular. Pulse radiolysis studies on GOase(semi) with CO2.- (which is unable To provide protons) are also described. These indicate that, after initial reduction to the Cu-I state (GOase(red)), a spontaneous decay of the unstable product occurs with formation of a disulfide radical anion (RSSR.-) detected by its absorbance at 450 nm. Slow decay of the disulfide radical is observed in a further step. Evidence obtained suggests that the spontaneous decay of the tyrosyl radical of GOase(ox) also involves the disulfide. As a means of modelling substrate binding, NCS- substitution at the Cu-II active site of GOase(ox) has been investigated. Dependence on pH is again observed and at 25 degrees C, pH 7.0 (10 mM phosphate), K = 0.5 x 10(3) M (-) (1), with forward rate constant k(f) = 1.1 x 10(4) M-1 s(-1), I = 0.100 M (NaCl). (C) 1999 Elsevier Science S.A. All rights reserved.