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A series of ascorbate derivatives has been used to examine the specificity of the reduction site of ascorbate oxidase. Replacement of the 6-OH group of ascorbic acid with either hydrogen or bromine does not alter the substrate activity significantly. 6-Amino-6-deoxy-L-ascorbic acid is a weak substrate for the enzyme, suggesting that positively charged groups at the 6-position are not well tolerated by the enzyme. The modification of the 5-OH reduces the effective interaction with the enzyme and the replacement of 6-OH with 6-S-phenyl- or 6-O-phenyl groups significantly increases the affinity for the enzyme. Both 2-Amino-6-S-phenyl-L-ascorbic acid and imino-D-glucoascorbic acid are not substrates for the enzyme. The stereoelectronic properties and alternate binding modes of these molecules are being considered to explain these observations. The substrate specificity of the enzyme is compared to the specificity of the reduction site of dopamine β-monooxygenase.

A series of ascorbate derivatives has been used to examine the specificity and the chemistry of the reduction site of dopamine β-monooxygenase (DβM). Replacement of the 6-OH group of ascorbic acid with either bromine or hydrogen does not alter the enzyme reduction efficiency significantly. Unexpectedly, the 6-OH modified ascorbate derivatives, 6-S-phenyl-6-thio-L-ascorbic acid and 6-O-phenyl-L-ascorbic acid were found to have much higher affinity for the enzyme than the most effective known electron donor, ascorbic acid (AscH−). The affinity of 2-amino-6-S-phenyl-L-ascorbic acid was found to be similar to that of 2-amino-L-ascorbic acid. 6-Amino-6-deoxy-L-ascorbic acid is neither a substrate nor an inhibitor for the enzyme. Although glucoascorbic acid is an excellent substrate for the enzyme, imino glucoascorbic acid was found to be an extremely potent competitive inhibitor for the enzyme. The stereoelectronic properties and alternate binding modes of these molecules have been considered in explaining the observations.

The interactions of reductants with dopamine beta-monooxygenase (DbetaM) were examined using two novel classes of reductants. The steady-state kinetics of the previously characterized DbetaM reductant, N,N-dimethyl-1,4-p-phenylenediamine (DMPD), were parallel to the ascorbic acid-supported reaction with respect to pH dependence and fumarate activation. DMPD also displayed pH and fumarate-dependent apparent negative cooperativity demonstrating that the previously reported cooperative behavior of DbetaM toward the reductant is not unique to ascorbic acid. The 6-OH phenyl and alkylphenyl-substituted ascorbic acid derivatives were more efficient reductants for the enzyme than ascorbic acid. Kinetic studies suggested that these derivatives behave as pseudo bisubstrates with respect to ascorbic acid and the amine substrate. The lack of apparent cooperative behavior with these derivatives suggests that this behavior of DbetaM is not common for all the reductants. Based on these findings and additional kinetic evidence, the proposal that the apparent negative cooperativity in the interaction of ascorbic acid with DbetaM was due to the presence of a distinct allosteric regulatory site has been ruled out. In contrast to previous models, where fumarate was proposed to interact with a distinct anion binding site, the effect of fumarate on the steady-state kinetics of these novel reductants suggests that fumarate and the reductant may interact with the same site of the enzyme. In accordance with these observations and mathematical analysis of the experimental data, a unified model for the apparent negative cooperativity and fumarate activation of DbetaM in which both fumarate and the reductant interact with the same site of all forms of the enzyme with varying affinities under steady-state turnover conditions has been proposed.

Recently, we reported the development of a sensitive continuous spectrophotometric assay for the ascorbate-dependent mammalian enzyme dopamine β-monooxygenase based on the novel chromophoric electron donor 2-aminoascorbic acid [K. Wimalasena and D. S. Wimalasena (1991) Anal. Biochem.197, 353-361]. We now report that ascorbate oxidase (EC 1.10.3.3, L-ascorbate:O2oxidoreductase) also catalyzes the oxidation of 2-aminoascorbic acid to chromophoric 2,2′-nitrilodi-2(2′)-deoxy-L-ascorbic acid (red pigment). The reaction is kinetically well behaved, displaying the expected stoichiometry for an oxidase-catalyzed reaction with respect to oxygen and the oxidation product (red pigment), demonstrating that 2-aminoascorbic acid is a well-behaved alternative substrate for the enzyme. Ascorbate oxidase is a very efficient enzyme toward its natural substrate, ascorbic acid. Although 2-aminoascorbic acid is a significantly weak substrate for the enzyme in comparison to ascorbic acid, as indicated by the apparent initial rate kinetic parameters, the high extinction coefficient of the red pigment under our assay conditions suggests that this novel reactivity of the enzyme could be used to design a sensitive, convenient, and continuous spectrophotometric assay for ascorbate oxidase. While this assay is more convenient than the existing oxygen monitor assay, its adaptability to measure the activity of the enzyme in the immobilized form may be helpful in the development of technologies for the automated detection of ascorbic acid in biological fluids for industrial or clinical applications. In addition, this novel reactivity of the enzyme may be used to examine the substrate specificity and the mechanism of action of the enzyme.