Galactose Oxidase as a Model for Reactivity at a Copper Superoxide Center

Abstract

The mononuclear copper enzyme, galactose oxidase, has been investigated under steady-state conditions via O₂-consumption assays using 1-O-methyl-α-d-galactopyranoside as the sugar substrate to produce an aldehyde at the C-6 position. The rate-determining step of the oxidative half-reaction was probed through the measurement of substrate and solvent deuterium and O-18 isotope effects on k_cat/K_m(O₂). The reaction conforms to a ping-pong mechanism with the kinetic parameters for the reductive half, k_cat/K_m(S) = 8.3 × 10³ M⁻¹ s⁻¹ at 10 °C and pH 7.0, comparing favorably to literature values. The oxidative half-reaction yielded a value of k_cat/K_m(O₂) = 2.5 × 10⁶ M⁻¹ s⁻¹. A substrate deuterium isotope effect of 32 was measured for the k_cat/K_m(S), while a smaller, but significant value of 1.6−1.9 was observed on k_cat/K_m(O₂). O-18 isotope effects of 1.0185 with either protiated or deuterated sugar, together with the absence of any solvent isotope effect, lead to the conclusion that hydrogen atom transfer from reduced cofactor to a Cu(II)−superoxo intermediate is fully rate-determining for k_cat/K_m(O₂). The measured O-18 isotope effects provide corroborative evidence for the reactive superoxo species in the dopamine β-monooxygenase/peptidylglycine α-hydroxylating monooxygenase family, as well as providing a frame of reference for copper−superoxo reactivity. The combination of solvent and substrate deuterium isotope effects rules out solvent deuterium exchange into reduced enzyme as the origin of the relatively small substrate deuterium isotope effect on k_cat/K_m(O₂). These data indicate fundamental differences in the hydrogen transfer step from the carbon of substrate vs the oxygen of reduced cofactor during the reductive and oxidative half-reactions of galactose oxidase.