Stimulation of Enzyme Activity by Ascorbate In Vitro

Over the years, a number of enzymes have been assumed to be ascorbate-dependent, because their activity is stimulated in vitro by the addition of ascorbate to the incubation medium. In general, these reactions are not ascorbate-dependent; ascorbate is one of a variety of reducing reagents that enhance the reaction.

Ascorbate is also frequently added to the incubation medium to remove hydrogen peroxide formed during a variety of reactions. Again, a number of reducing agents have the same action, and in vivo this role would presumably be performed by catalase.

In vitro, ascorbate and Fe2+ ions are frequently used as a source of superoxide for such enzymes as indoleamine dioxygenase. Although ascorbate does have prooxidant and superoxide generating activity (Section 13.3.7), there is no evidence that it is the physiological source of this radical for superoxide-utilizing enzymes.

There is a long-standing myth that ascorbate is required for the hydroxy-lation of tyrosine to dihydroxyphenylalanine (see Figure 13.4) and the similar reactions of phenylalanine and tryptophan hydroxylases. This belief arose as a result of early studies of a nonenzymic reaction to synthesize the hydroxy-lated amino acids for further study. It became established that ascorbate was required for these hydroxylations, and it is still common to include it in the incubation buffer. So far from requiring ascorbate, the addition of relatively low concentrations of ascorbate to preparations of tyrosine hydroxylase that has been activated by cAMP-dependent protein kinase results in irreversible loss of activity, although the unactivated form of the enzyme is unaffected by ascorbate (Wilgus and Roskoski, 1988). As discussed in Section 10.4.1, these enzymes are biopterin-dependent, and require dihydrobiopterin reductase and NADPH for activity. There is, however, evidence that, in some nerve cell lines in culture, tyrosine hydroxylase may be induced by ascorbate (Seitz et al., 1998).

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