Purification and characterization of recombinant human liver glycolate oxidase.
Glycolate oxidase, an FMN-dependent peroxisomal oxidase, plays an important role in plants, related to photorespiration, and in animals, where it can contribute to the production of oxalate with formation of kidney stones. The best studied plant glycolate oxidase is that of spinach; it has been expressed as a recombinant enzyme, and its crystal structure is known. With respect to animals, the enzyme purified from pig liver has been characterized in detail in terms of activity and inhibition, the enzyme from human liver in less detail. We describe here the purification and initial characterization of the recombinant human glycolate oxidase. Its substrate specificity and the inhibitory effects of a number of anions are in agreement with the properties expected from previous work on glycolate oxidases from diverse sources. The recombinant enzyme presents an inhibition by excess glycolate and by excess DCIP, which has not been documented before. These inhibitions suggest that glycolate binds to the active site of the reduced enzyme, and that DCIP also has affinity for the oxidized enzyme. Glycolate oxidase belongs to a family of l-2-hydroxy-acid-oxidizing flavoenzymes, with strongly conserved active-site residues. A comparison of some of the present results with studies dealing with other family members suggests that residues outside the active site influence the binding of a number of ligands, in particular sulfite.
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS FRE2930, 91198 Gif-sur-Yvette Cedex, France.
Caroline
C
Vignaud
Nicolas
N
Pietrancosta
Emma L
EL
Williams
Gill
G
Rumsby
Florence
F
Lederer
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004789:Enzyme Activation; D004795:Enzyme Stability; D006801:Humans; D008099:Liver / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C020821:glycollate oxidase