Experimental Evidence for a Hydride Transfer Mechanism in Plant Glycolate Oxidase Catalysis

• Published in: The Journal of biological chemistry 2015-01, Vol.290 (3), p.1689-1698
• Main Authors: Dellero, Younès, Mauve, Caroline, Boex-Fontvieille, Edouard, Flesch, Valérie, Jossier, Mathieu, Tcherkez, Guillaume, Hodges, Michael
• Format: Article
• Language: English
• Subjects: Alcohol Oxidoreductases - metabolism; Arabidopsis - enzymology; Carbon Isotopes - chemistry; Catalysis; Deuterium - chemistry; Enzyme; Enzymology; Escherichia coli - metabolism; Glycolate Oxidase; Hydride Transfer; Isotope Effect; Life Sciences; Light; Models, Chemical; Oxidase; Photorespiration; Plant; Plant Extracts - chemistry; Plant Leaves - metabolism; Plant Physiological Phenomena; Plant Proteins - metabolism; Protein Binding; Protein Conformation; Solvents - chemistry; Zea mays - enzymology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.618629

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 12C/13C isotope effect was also investigated for each plant glycolate oxidase protein by measuring the 13C 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.Uncertainty remains about the nature of transition states along the reductive half-reaction of glycolate oxidase. Deuterated glycolate and solvent slow down plant glycolate oxidase catalysis to a modest extent. Isotope effects support a hydride transfer mechanism and indicate glycolate deprotonation to be only partially rate-limiting. Understanding the catalytic mechanism of the enzyme is crucial for designing drugs/herbicides to inhibit its activity.