Solvent and solvent proton dependent steps in the galactose oxidase reaction

Publisher Website
Google Scholar
Abstract
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 .beta.-galactopyranoside by O2 was measured. Examination of the thermodynamic activation parameters for the reaction indicated that the isotope effect was attributable to a slightly less favorable .DELTA.H value, consistent with a KSIE on proton transfer. A detailed kinetic analysis was performed, examining the effect of D2O on the rate of reaction over the pH range 4.8-8.0. Both pL-rate profiles exhibited bell-shaped curves. Substitution of D2O as solvent shifted the pKes values for the enzymic central complex: pKes1 from 6.30 to 6.80 and pKes2 from 7.16 to 7.35. Analysis of the observed shifts in dissociation constants was performed with regard to potential hydrogenic sites. pKes1 can be attributed to a histidine imidazole, while pKes2 is tentatively assigned to a Cu2+-bound water moleculae. A proton inventory was performed (KSIE = +1.55); the plot of .kappa.cat vs. mole fraction D2O 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 Ki-pH profile indicated that a group ionization, with pKa = 7.17, modulated CN- inhibition; Ki 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 D2O-dependent shift in pKa (+0.17) of the group modulating CN- inhibition which was similar to the shift observed in pKes2. 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., and Kurland, R. J. (1981) J. Am. Chem. Soc. 103, 2842-2847]. The data indicate that this Cu(II)-bound H2O 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 O2 with the proton transfer step(s) probed by these experiments is discussed.