Circular dichroism spectra of the copper enzyme, galactose oxidase, in the presence of its substrates and products

Circular dichroism spectra were recorded for galactose oxidase in the presence of its substrates and products to determine which of these ligands interacts with the copper atom of the enzyme. Neither galactose nor oxygen has any detectable effect on protein conformation as evidenced by near-and far-ultraviolet circular dichroism spectra. In the absence of oxygen, D-galactose at concentrations an order of magnitude higher than its K_m causes a marked reduction of galactose oxidase copper optical activity. At the 314-and 610-nm copper extrema, the ellipticity is reduced to less than half of its value for the enzyme alone. On the other hand, removal or addition of oxygen to galactose oxidase solutions results in only small, barely significant, changes in copper optical activity which are in the same direction. In view of circular dichroism, kinetic, and electron spin resonance results, it is inferred that during the course of the enzyme reaction oxygen binds after galactose and interacts directly with reducing equivalents on the substrate rather than within the inner coordination sphere of the copper atom. In a manner similar to galactose, the aldehyde product of the enzyme reaction also interacts with the copper atom as evidenced by large changes in optical activity. Experiments with the other product, hydrogen peroxide, were somewhat ambiguous since this compound has effects on protein conformation in addition to its effects on copper optical activity. In addition to these effects on copper optical activity, large changes in nearultraviolet circular dichroism spectra are observed with dihydroxyacetone and galactohexodialdose. These changes indicate that a tryptophan(s) residue(s) is at or near the active site of galactose oxidase.