Expression of Spinach Glycolate Oxidase in Saccharomyces cerevisiae: Purification and Characterization

Glycolate oxidase from spinach has been expressed in Saccharomyces cerevisiae. The active enzyme was purified to near-homogeneity (purification factor ~ 1400-fold) by means of hydroxyapatite and anion-exchange chromatography. The purified glycolate oxidase is nonfluorescent and has absorbance peaks at 448 (∈ = 9200 M⁻¹ cm⁻¹) and 346 nm in 0.1 M phosphate buffer, pH 8.3. The large bathochromic shift of the near-UV band indicates that the N(3) position is deprotonated at pH 8.3. A pH titration revealed that the pK of the N(3) is shifted from 10.3 in free flavin to 6.4 in glycolate oxidase. Glycolate oxidase is competitively inhibited by oxalate with a K_d of 0.24 mM at 4 °C in 0.1 M phosphate buffer, pH 8.3. Three pieces of evidence demonstrate that glycolate oxidase stabilizes a negative charge at the N(l)-C(2=0) locus: the enzyme forms a tight sulfite complex with a K_d of 2.7 × 10⁻⁷ M and stabilizes the anionic flavosemiquinone and the benzoquinoid form of 8-mercapto-FMN. Steady-state analysis at pH 8.3, 4 °C, yielded a K_m = 1 × 10⁻³ M for glycolate and K_m = 2.1 × 10⁻⁴ M for oxygen. The turnover number has been determined to be 20 s⁻¹. Stopped-flow studies of the reductive (k = 25 s⁻¹) and oxidative (k = 8.5 X 10⁴ M⁻¹ s⁻¹) half-reactions have identified the reduction of glycolate oxidase to be the rate-limiting step.