Thiols as mechanistic probes for catalysis by the free radical enzyme galactose oxidase.
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 redox 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-bonding interactions due to the larger atomic radius of sulfur compared to that of oxygen.
Wachter
R M
RM
Department of Chemistry, University of Oregon, Eugene 97403, USA.
Branchaud
B P
BP
D001665:Binding Sites; D002384:Catalysis; D003300:Copper / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry; D005609:Free Radicals; D005670:Fusarium / Q000201:enzymology; D005692:Galactose Oxidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D006862:Hydrogen Sulfide / Q000737:chemistry; D010084:Oxidation-Reduction; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D004791:Enzyme Inhibitors; D005609:Free Radicals; D013438:Sulfhydryl Compounds; D003300:Copper; D005692:Galactose Oxidase; D006862:Hydrogen Sulfide