Kinetic and magnetic resonance studies of substrate binding to galactose oxidase copper(II)

Abstract

Alcohol substrate binding to the copper-containing enzyme galactose oxidase (GOase) has been studied by kinetic competition against cyanide and fluoride, 13C nmr relaxation, and esr competition experiments. The 13C nmr spectra of the substrate β-O-methyl- d-galactopyranoside (β-O-me-gal) show no apparent paramagnetic relaxation rate enhancement that could be attributed to innersphere equatorial binding of this molecule at the Cu(II) center. Moreover, the kinetics observed when CN − or F − are used as inhibitors of GOase with β-O-me-gal as the substrate suggest that these anions act as apparent noncompetitive inhibitors; the binding of the substrates β-O-me-gal and O 2 is not hindered per se, but the catalytic activity of the enzyme substrate complex is greatly decreased. The esr competition data also confirm that, in the absence of O 2, CN − and β-O-me-gal do not compete for the same GOase binding site. Previously reported esr and 19F nmr data show that CN − binds to the GOase Cu(II) at an equatorial coordination site, as does the F − detected in esr experiments. Thus, the results from the various competition experiments support a model in which alcohol substrates bind outersphere to the GOase Cu(II), or, possibly, to an axial site.