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

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 C 3 and C 4 plant species, we analyzed kinetic parameters of purified recombinant C 3 ( Arabidopsis thaliana ) and C 4 ( 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 C 3 and C 4 enzymes.Uncertainty remains about the nature of transition states along the reductive half-reaction of glycolate oxidase. Results Deuterated glycolate and solvent slow down plant glycolate oxidase catalysis to a modest extent. Conclusion Isotope effects support a hydride transfer mechanism and indicate glycolate deprotonation to be only partially rate-limiting. Significance Understanding the catalytic mechanism of the enzyme is crucial for designing drugs/herbicides to inhibit its activity.