ABSTRACT
Glycolate oxidase (GO) was purified to homogeneity from leaves of spinach (Spinacia oleracea). Through detecting the consumption of oxygen and the formation of hydrogen peroxide in the assay solution, it was found that GO could also oxidize glycerate, another metabolite in the photorespiratory pathway, and use FMN and FAD, but not riboflavin and lumiflavin, as its cofactors. The optimum reaction pH, Km for glycerate, k(cat) and activation energy of this oxidizing reaction were determined to be 8.0, 7.14 mmol/L, 1.04 s(-1) and 17.29 kJ/mol, respectively. Oxalate and pyruvate at 5.0 mmol/L could inhibit the glycerate-oxidizing activity by 34% and 26%, and oxalate acted as a competitive inhibitor of the glycerate oxidation reaction with a K(i) of 0.75 mmol/L. By the competition plotting with mixed-substrates, it was indicated that glycolate-oxidizing activity and glycerate-oxidizing activity of GO shared the same active site.
Subject(s)
Alcohol Oxidoreductases/metabolism , Glyceric Acids/metabolism , Plant Leaves/enzymology , Plant Proteins/metabolism , Spinacia oleracea/enzymology , Hydrogen-Ion Concentration , Kinetics , Oxidation-Reduction , Plant Leaves/metabolism , Spinacia oleracea/metabolismABSTRACT
The dependence of glycolate oxidase apoprotein (apoGO) activity on flavin analogs was surveyed in 9 higher plants from 7 families. Activities of all apoGOs depended not only on flavin mononucleotide (FMN) but also on flavin adenine dinucleotide (FAD), but not on riboflavin. The kinetic analysis showed that FMN was the optimum cofactor for apoGO from leaves of Brassica campestris. In plant kingdom, FMN, FAD and riboflavin are three flavin analogs with very similar structure, and they could coexist and be inter-converted from each other, so the question is how the apoprotein of glycolate oxidase (GO) recognized these flavin analogs. No inhibition effect of riboflavin on the activity of apoGO with FMN or FAD was found and no obvious quenching of riboflavin or apoGO protein fluorescence was detected with the addition of apoGO or riboflavin, respectively. These results indicated that riboflavin did not bind to apoGO tightly like FMN and FAD. Inorganic phosphate (Pi) did inhibit the activity of GO, and kinetic analysis revealed that this inhibition was caused by the competitive binding to apoGO between Pi and FMN. This competitive binding was further confirmed by the inhibition of Pi to the quenching of FMN and apoGO protein fluorescence with apoGO and FMN, respectively. It was suggested that the 5'-phosphate group of FMN or FAD may play a key role in the recognition and binding of riboflavin analog cofactors with apoGO.
