Galactose oxidase, a mononuclear copper enzyme, oxidizes primary alcohols to aldehydes using molecular oxygen. A unique type of cross-link between tyrosine 272, an active site copper ligand, and cysteine 228 provides a modified tyrosine radical site believed to act as a one-electron redox center. Substrate analogs incorporating a primary thiol group in place of the primary alcohol group in normal substrates (RCH2OH) have been studied as active-site mechanistic probes. Thiol sulfur coordinates to the active-site copper, leading to enzyme inactivation in a time- and concentration-dependent manner. The mechanism of inactivation involves fedex chemistry related to the active-site redox centers, though inactivation does not proceed through the rate-determining hydrogen atom abstraction step that occurs in alcohol oxidation. Thiols are therefore classified as active site-directed redox inactivators. The thiol analog of galactose, 6-Thio-Me-Gal, is also turned over by the enzyme, albeit at a much reduced rate, indicating that the energetics of turnover is changed significantly. Thiols constitute a particularly good model of the ground state enzyme-substrate complex. The Michaelis complex for thiol substrate analogs is stabilized at least 200- fold compared to the analogous alcohol substrates, whereas the transition state of H atom abstraction is destabilized, presumably due to a slight increase in distances of reacting atoms and weakening of hydrogen-binding interactions due to the larger atomic radius of sulfur compared to that of oxygen.
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