Kinetic mechanism of the Cu(II) enzyme galactose oxidase

The steady-state kinetics of four redox reactions catalyzed by galactose oxidase have been determined. The alcohol substrate used in each case was galactose; the four oxidant substrates used were O 2, IrCl 6 2−, porphyrexide, and Fe(CN) 6 3−. With the exception of the last reagent, saturation behavior is exhibited by all substrates. Double reciprocal plots of rate data obtained varying one substrate at various concentrations of the other are intersecting for all pairs that exhibited saturation behavior. Thus, these reactions are kinetically sequential processes involving single central complexes. These complexes involve enzyme, galactose, and one molecule of oxidant, whether or not the oxidant is a one- or two-electron acceptor. This result indicates that for one-electron oxidants, an enzyme-alcohol-derived radical species may exist as a transient prior to the reaction of the second electron equivalent of oxidant. A similar substrate ▪ transient is postulated in the reaction involving O 2. The inhibition by H 2O 2 has also been studied in detail. H 2O 2 apparently binds to the enzyme at two sites. The nature of alcohol and O 2 binding to the enzyme Cu(II) is discussed in light of these kinetic results.