Ascorbate Oxidase

Highly purified ascorbate oxidase has several characteristics not reported previously. The monoanion is the active form of the reductant substrate. The rate-limiting reaction in the catalytic cycle at ascorbate saturation requires protonation of a negatively charged enzymic group with a pK′ near pH 8. Although citrate buffers are commonly used for assay of this enzyme, the univalent citrate anion is a competitive inhibitor with respect to ascorbate. Piperazine-N,N′-bis(2-ethanesulfonic acid) anions also inhibit competitively but EDTA has no effect except to prevent the nonenzymic oxidation of ascorbate. The maximal velocity of the isolated enzyme can be increased severalfold by treatment with excess ascorbate in the absence of oxygen. This activation process may involve generation of additional catalytically competent sites.