title: Experimental evidence for a hydride transfer mechanism in plant glycolate oxidase catalysis.
abstract: In plants, glycolate oxidase is involved in the photorespiratory cycle, one of the major fluxes at the global scale. To clarify both the nature of the mechanism and possible differences in glycolate oxidase enzyme chemistry from C3 and C4 plant species, we analyzed kinetic parameters of purified recombinant C3 (Arabidopsis thaliana) and C4 (Zea mays) plant enzymes and compared isotope effects using natural and deuterated glycolate in either natural or deuterated solvent. The (12)C/(13)C isotope effect was also investigated for each plant glycolate oxidase protein by measuring the (13)C natural abundance in glycolate using natural or deuterated glycolate as a substrate. Our results suggest that several elemental steps were associated with an hydrogen/deuterium isotope effect and that glycolate α-deprotonation itself was only partially rate-limiting. Calculations of commitment factors from observed kinetic isotope effect values support a hydride transfer mechanism. No significant differences were seen between C3 and C4 enzymes.
authors.affiliation: From the Institut de Biologie des Plantes, CNRS UMR8618, Saclay Plant Sciences, Bâtiment 630, Université Paris Sud, 91405 Orsay Cedex, France.
authors.forename: Younès
authors.initials: Y
authors.lastname: Dellero
authors.affiliation: From the Institut de Biologie des Plantes, CNRS UMR8618, Saclay Plant Sciences, Bâtiment 630, Université Paris Sud, 91405 Orsay Cedex, France, Plateforme Métabolisme-Métabolome, Saclay Plant Sciences, Institut de Biologie des Plantes, Bâtiment 630, 91405 Orsay Cedex, France, and.
authors.forename: Caroline
authors.initials: C
authors.lastname: Mauve
authors.affiliation: From the Institut de Biologie des Plantes, CNRS UMR8618, Saclay Plant Sciences, Bâtiment 630, Université Paris Sud, 91405 Orsay Cedex, France.
authors.forename: Edouard
authors.initials: E
authors.lastname: Boex-Fontvieille
authors.affiliation: From the Institut de Biologie des Plantes, CNRS UMR8618, Saclay Plant Sciences, Bâtiment 630, Université Paris Sud, 91405 Orsay Cedex, France.
authors.forename: Valérie
authors.initials: V
authors.lastname: Flesch
authors.affiliation: From the Institut de Biologie des Plantes, CNRS UMR8618, Saclay Plant Sciences, Bâtiment 630, Université Paris Sud, 91405 Orsay Cedex, France.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Jossier
authors.affiliation: From the Institut de Biologie des Plantes, CNRS UMR8618, Saclay Plant Sciences, Bâtiment 630, Université Paris Sud, 91405 Orsay Cedex, France, Plateforme Métabolisme-Métabolome, Saclay Plant Sciences, Institut de Biologie des Plantes, Bâtiment 630, 91405 Orsay Cedex, France, and Institut Universitaire de France, 103 Boulevard Saint-Michel, 75005 Paris, France.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Tcherkez
authors.affiliation: From the Institut de Biologie des Plantes, CNRS UMR8618, Saclay Plant Sciences, Bâtiment 630, Université Paris Sud, 91405 Orsay Cedex, France, michael.hodges@u-psud.fr.
authors.forename: Michael
authors.initials: M
authors.lastname: Hodges
mesh_terms: D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology*; D002247:Carbon Isotopes / Q000737:chemistry; D002384:Catalysis; D003903:Deuterium / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D008027:Light; D008956:Models, Chemical; D010936:Plant Extracts / Q000737:chemistry; D018515:Plant Leaves / Q000378:metabolism; D018521:Plant Physiological Phenomena; D010940:Plant Proteins / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012997:Solvents / Q000737:chemistry; D003313:Zea mays / Q000201:enzymology*
chemical_list: D002247:Carbon Isotopes; D010936:Plant Extracts; D010940:Plant Proteins; D012997:Solvents; D003903:Deuterium; D000429:Alcohol Oxidoreductases; C020821:glycollate oxidase
keywords: Catalysis; Enzyme; Glycolate Oxidase; Hydride Transfer; Isotope Effect; Oxidase; Photorespiration; Plant