title: 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 beta-O-methyl-D-galactopyranoside (beta-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 beta-O-me-gal as the substrate suggest that these anions act as apparent non-competitive inhibitors; the binding of the substrates beta-O-me-gal and O2 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 O2, CN- and beta-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 supports a model in which alcohol substrates bind outersphere to the GOase Cu(II), or, possibly, to an axial site.
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D003300:Copper; D003486:Cyanides / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D005459:Fluorides / Q000494:pharmacology; D005692:Galactose Oxidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008667:Metalloproteins / Q000378:metabolism; D008756:Methylgalactosides / Q000378:metabolism*; D008759:Methylglycosides / Q000378:metabolism*; D003904:Mitosporic Fungi / Q000201:enzymology*
chemical_list: D003486:Cyanides; D008667:Metalloproteins; D008756:Methylgalactosides; D008759:Methylglycosides; C031552:methyl beta-galactoside; D003300:Copper; D005692:Galactose Oxidase; D005459:Fluorides