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 approximately 1400-fold) by means of hydroxyapatite and anion-exchange chromatography. The purified glycolate oxidase is nonfluorescent and has absorbance peaks at 448 (epsilon = 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 Kd of 0.24 mM at 4 degrees 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(1)-C(2 = O) locus: the enzyme forms a tight sulfite complex with a Kd of 2.7 x 10(-7) M and stabilizes the anionic flavosemiquinone and the benzoquinoid form of 8-mercapto-FMN. Steady-state analysis at pH 8.3, 4 degrees C, yielded a Km = 1 x 10(-3) M for glycolate and Km = 2.1 x 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 10(4) M-1 s-1) half-reactions have identified the reduction of glycolate oxidase to be the rate-limiting step.
Macheroux
P
P
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
Massey
V
V
Thiele
D J
DJ
Volokita
M
M
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004795:Enzyme Stability; D005486:Flavin Mononucleotide / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006882:Hydroxyapatites; D007700:Kinetics; D010944:Plants / Q000201:enzymology*; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014170:Transformation, Genetic
D006882:Hydroxyapatites; C068726:8-mercapto-FMN; D005486:Flavin Mononucleotide; D017886:Durapatite; D000429:Alcohol Oxidoreductases; C020821:glycollate oxidase