Thiols as mechanistic probes for catalysis by the free radical enzyme galactose oxidase

Rebekka M. Wachter, Bruce Branchaud

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)14425-14435
Number of pages11
JournalBiochemistry
Volume35
Issue number45
DOIs
StatePublished - Nov 26 1996
Externally publishedYes

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Galactose Oxidase
Catalysis
Sulfhydryl Compounds
Free Radicals
Catalytic Domain
Enzymes
Alcohols
Oxidation-Reduction
Substrates
Copper
Sulfur
Atoms
Hydrogen
Oxygen
Molecular oxygen
Galactose
Aldehydes
Ground state
Cysteine
Tyrosine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Thiols as mechanistic probes for catalysis by the free radical enzyme galactose oxidase. / Wachter, Rebekka M.; Branchaud, Bruce.

In: Biochemistry, Vol. 35, No. 45, 26.11.1996, p. 14425-14435.

Research output: Contribution to journalArticle

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