Studies on Glycollate Oxidase from Pea Leaves : Determination of Stereospecificity and Mode of Inhibition by alpha-Hydroxybutynoate

Glycollate oxidase (glycollate:oxygen oxidoreductase, EC 1.1.3.1) from Pisum sativum has an unusual absorption spectrum which suggests that the flavin N(3)-H function of the FMN coenzyme is ionized at pH 8.3. The enzyme is reduced rapidly by the substrate glycollate to yield a normal, reduced FMN coenzyme which is readily reoxidized by O2. No evidence for the occurrence of covalent intermediates during reduction, as observed upon reduction of L-Iactate oxidase from Mycobacterium smegmatis with the same substrate (Ghisla, S. and Massey, V. (1980) J. BioI. Chem. 255, 5688-5696), could be obtained. The enzyme was determined to be greater than 99.5% specific in the abstraction of the Re-hydrogen of glycollate. D-Lactate dehydrogenase from Lactobacillus Jeichmanii was shown to be only 97% selective for the Si-side in the reduction of glyoxylate. Glycollate oxidase was shown to be inhibited by α-hydroxybutynoate via covalent modification of the FMN coenzyme, in a fashion similar to that encountered with L-Iactate oxidase (Schonbrunn, A., Abeles, R.H., Walsh, Ch.T., Ghisla, S., Ogata, H. and Massey, V. (1976) Biochemistry 15, (1798-1807). This inhibitor serves both as substrate and inactivator of the enzyme