Solvent and Solvent Proton Dependent Steps in the Galactose Oxidase Reaction

Solvent and solvent proton dependent steps involved in the mechanism of the enzyme galactose oxidase have been examined. The deuterium kinetic solvent isotope effect (KSIE) on the velocity of the galactose oxidase catalyzed oxidation of methyl ß-galactopyranoside by 0₂ was measured. Examination of the thermodynamic activation parameters for the reaction indicated that the isotope effect was attributable to a slightly less favorable value, consistent with a KSIE on proton transfer. A detailed kinetic analysis was performed, examining the effect of D₂0 on the rate of reaction over the pH range 4.8-8.0. Both pL-rate profiles exhibited bell-shaped curves. Substitution of D₂0 as solvent shifted the values for the enzymic central complex: from 6.30 to 6.80 and from 7.16 to 7.35. Analysis of the observed shifts in dissociation constants was performed with regard to potential hydrogenic sites. can be attributed to a histidine imidazole, while is tentatively assigned to a Cu²⁺-bound water molecule. A proton inventory was performed (KSIE = +1.55); the plot of K_cat vs. mole fraction D₂0 was linear, indicating the existence of a single solvent-derived proton involved in a galactose oxidase rate-determining step (or steps). The pH dependence of CN^-inhibition was also examined. The profile indicated that a group ionization, with pK_a = 7.17, modulated CN^-inhibition; K_i was at a minimum when this group was in the protonated state. The inhibition profile followed the alkaline limit of the pH-rate profile for the enzymic reaction, suggesting that the group displaced by CN^-was also deprotonating above pH 7. Consistent with this suggestion was the D₂0-dependent shift in (+0.17) of the group modulating CN^-inhibition which was similar to the shift observed in Nuclear and electron magnetic resonance studies have shown previously that CN^-coordinates equatorially to the enzymic Cu(II), apparently displacing a water molecule [Marwedel, B. J., Kosman, D.J., Bereman, R. D., & Kurland, R. J. (1981) J. Am. Chem. Soc. 103, 2842–2847]. The data indicate that this Cu(II)-bound H₂0 is required in the protonated aquo state for catalysis and is responsible for the KSIE observed in the pL-rate profile. A mechanism that couples electron transfer to 0₂ with the proton transfer step(s) probed by these experiments is discussed.