Dopamine hydroxylase substrate specificity

Dopamine -hydroxylase is a copper-containing enzyme involved in the synthesis of the catecholamines noradrenaline and adrenaline from tyrosine in the adrenal medulla and central nervous system (see Figure 13.4). The active enzyme contains Cu+, which is oxidized to Cu + during the hydroxylation of the substrate. Reduction back to Cu+ specifically requires ascorbate, which is oxidized to monodehydroascorbate. 

Dopamine -hydroxylase catalyzes the side-chain hydrox-ylation of dopamine and other phenylethylamine derivatives. Ascorbic acid serves as a specific electron-donating cofactor. The enzyme from bovine adrenal glands contains and a smaller amount of Cu. When the enzyme oxidizes ascorbate to dehydroascorbate, most of the is reduced to Cu Added substrate is hydroxylated, and Cu is reoxidized to Cu This indicates that most of the protein-bound Cu undergoes cyclic reduction and oxidation during hydroxyhtion. The results also rule out an oxygen-carrier function for ascorbate. The possibility that a p-substituted hydroperoxide of the substrate is formed as an intermediate in the reaction has been examined with the use of 13,13 -tritium-labelled substrate. The results indicate that such an intermediate is unlikely. 

The final stage in the biosynthesis of NA involves a second mixed function oxidase, dopamine- -hydroxylase. This enzyme has been extensively purified from the bovine adrenal medulla, and is a copper-containing protein of molecular weight 290,000. Ascorbic acid acts as the cofactor (XHj). Enzyme activity is stimulated by catalytic amounts of fumaric acid and by certain other dicarboxylic acids. The enzyme has a broad substrate specificity, and will catalyse the /7-hydroxylation of a large number of phenylethylamine derivatives, including a number of sympathomimetic amines (Table 8). 

SYNTHESIS, STORAGE, AND RELEASE OF CATECHOLAMINES Synthesis—The steps in the synthesis of DA, NE (known outside the U.S. as noradrenaline), and Epi (known as adrenahne) are shown in Eigure 6-A. Tyrosine is sequentially 3-hydroxylated and decarboxylated to form DA. DA is 3-hydroxylated to yield NE (the transmitter in postganglionic nerves of the sympathetic branch of the ANS), which is N-methylated in chromaffin tissue to give Epi. The enzymes involved are not completely specific consequently, other endogenous substances and some drugs are also substrates. 5-hydroxytryptamine (5-HT, serotonin) can be produced from 5-hydroxy-L-tryptophan by aromatic L-amino acid decarboxylase (AAD or dopa decarboxylase). AAD also converts dopa into DA, and methyldopa to a-methyl-DA, which is converted to a-methyl-NE by dopamine /3-hydroxylase (Dj3H Table 6-4). 

Reactions 2, 3, and 4 tell us little about how oxygen is activated during the hydroxylation reaction, and at the moment one can only speculate about the details. The scheme and the results on which it is based do, however, rule out several general types of hydroxylation mechanism. The fact that the enzyme can be reduced anaerobically by ascorbate to a form which actively supports substrate hydroxylation in the absence of ascorbate rules out any mechanism for this enzyme-catalyzed reaction in which the ascorbate functions as an oxygen carrier. Such a role has been postulated for tetrahydropteridines (id), which can serve as specific electron-donating cofactors (just as ascorbate does with dopamine )S-hydroxylase) in certain aromatic hydroxylation reactions (iO, 12). 

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