THIOLS AS MECHANISTIC PROBES FOR CATALYSIS BY THE FREE-RADICAL ENZYMEGALACTOSE-OXIDASE

Galactose oxidase, a mononuclear copper enzyme, oxidizes primary alcoh ols to aldehydes using molecular oxygen. A unique type of cross-link b etween tyrosine 272, an active-site copper ligand, and cysteine 228 pr ovides a modified tyrosine radical site believed to act as a one-elect ron redox center. Substrate analogs incorporating a primary thiol grou p in place of the primary alcohol group in normal substrates (RCH(2)OH ) have been studied as active-site mechanistic probes, Thiol sulfur co ordinates to the active-site copper, leading to enzyme inactivation in a time- and concentration-dependent manner. The mechanism of inactiva tion involves redox chemistry related to the active-site redox centers , though inactivation does not proceed through the rate-determining hy drogen atom abstraction step that occurs in alcohol oxidation. Thiols are therefore classified as active-site-directed redox inactivators. T he thiol analog of galactose, 6-Thio-Me-Gal, is also turned over by th e enzyme, albeit at a much reduced rate, indicating that the energetic s of turnover is changed significantly. Thiols constitute a particular ly good model of the ground state enzyme-substrate complex. The Michae lis complex for thiol substrate analogs is stabilized at least 200-fol d compared to the analogous alcohol substrates, whereas the transition state of H atom abstraction is destabilized, presumably due to a slig ht increase in distances of reacting atoms and weakening of hydrogen-b onding interactions due to the larger atomic radius of sulfur compared to that of oxygen.