Fluoride ion as an NMR relaxation probe of galactose oxidase—substrate binding

Abstract

From the dependence on substrate concentration of fluoride ion spin-lattice and spin-spin paramagnetic relaxation rate enhancements, a value for the dissociation constant, K d = 0.059 ± 0.002 M, for the anaerobic binding of dihydroxyacetone (monomer) to the Cu(II) site of the enzyme galactose oxidase ( d-galactose:oxygen 6-oxidoreductase, EC 1.1.3.9) has been obtained. This value for K d lies between previously reported values for K m derived by use of classical Michaelis-Menten kinetics. An analogous calculation for the anaerobic binding of galactose to the enzyme yields K d = 0.145 ± 0.004 M, a value different from several reported Michaelis constants. F − NMR relaxation measurements on air-exposed samples of galactose and the enzyme yield a dissociation constant for the active site-oxidation product (presumed to be galactohexodialdose), K d = 2.2 ± 0.2 M, a value at least an order of magnitude larger than the Michaelis or dissociation constants calculated for the binding of galactose to the enzyme active site; no value for this constant had been reported previously. Some implications of the competition results for the type of substrate binding are discussed.