Purification and characterization of glycolate oxidase from pumpkin cotyledons.

Glycolate oxidase was purified and crystallized from cotyledons of germinating pumpkin seedlings. The molecular weight of the enzyme was determined to be 280,000-320,000, consisting of 8 identical subunits with molecular weight of 38,000. There are two absorption peaks at 340 and 450 nm, indicating the glycolate oxidase is a flavin protein. Several kinetic parameters were determined, Km (glycolate) 0.33 mM and Km (O2) 76.2 microM at pH 8.0. Oxalate and oxalacetate were found to be potent competitive inhibitors against glycolate; the Ki values for oxalate and oxalacetate were 4.5 and 7.8 mM, respectively. Fatty acids such as linoleic acid inhibited the enzyme noncompetitively; the Km for linoleic acid was 0.63 mM. The regulation of glycolate oxidase in the glycolate pathway occurring in leaf peroxisomes is discussed.