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Enzymatic iodination of tyrosine and thyroglobulin with chloroperoxidase.

A. Taurog,E. M. Howells

Published 1966 in Journal of Biological Chemistry

ABSTRACT

Abstract 1. Crystalline chloroperoxidase is effective in catalyzing the iodination of tyrosine or thyroglobulin when supplemented with H2O2, or with the H2O2-generating system, glucose-glucose oxidase. The iodination reaction is very rapid at low concentrations of iodide (5 x 10-5 m), and at low concentrations of tyrosine (1 x 10-4 m) or thyroglobulin (0.33 mg per ml). 2. Iodide is rapidly bound as 3-iodotyrosine, 3,5-diiodotyrosine, and thyroxine during the chloroperoxidase-catalyzed iodination. After 60 min of incubation with thyroglobulin as acceptor, chromatography of a Pronase digest showed approximately 45 to 50% of the added 131I in the form of 3,5-diiodotyrosine, about 20 to 25% as 3-iodotyrosine, and 4 to 5% as thyroxine. 3. Since iodination of thyroglobulin occurred very effectively in the presence of only a single crystalline enzyme, the results suggest that it may not be necessary to postulate the existence of a separate "tyrosine iodinase" in the thyroid. Moreover, the formation of appreciable thyroxine in the chloroperoxidase-catalyzed iodination of thyroglobulin suggests that a peroxidase may also be involved in the coupling reaction in the thyroid. 4. The chloroperoxidase-catalyzed iodination of tyrosine and thyroglobulin is greatly accelerated by 0.1 m chloride and bromide. The mechanism of this stimulatory effect is not yet known, although several possibilities are discussed. 5. Iodination of thyroglobulin and tyrosine in the presence of chloroperoxidase is readily inhibited by low concentrations of antithyroid drugs and by naturally occurring reducing agents such as cysteine, reduced glutathione, and ascorbic acid. Results obtained with these inhibitors support the view that antithyroid compounds act as competitive inhibitors in the formation of organic iodine in the thyroid. 6. Excess iodide inhibits the chloroperoxidase-catalyzed iodination of tyrosine and thyroglobulin. These results suggest that the antithyroid effect of iodide, observed under certain conditions, may be partly due to inhibition of thyroid peroxidase. 7. Chloroperoxidase is very effective in catalyzing iodination of insulin and serum albumin. 3,5-Diiodotyrosine formation was at least as rapid in the case of these acceptors as it was with thyroglobulin, but thyroxine formation was not as marked. These results suggest that steric arrangement of newly formed 3,5-diiodotyrosine residues in thyroglobulin was more favorable than in the other proteins for promoting the coupling reaction. 8. No evidence was obtained that 3,5-diiodo-4-hydroxyphenylpyruvic acid (DHP) is an intermediate in the formation of thyroxine during the iodination of thyroglobulin with chloroperoxidase. However, when free tyrosine was iodinated with chloroperoxidase, evidence was obtained that DHP was involved as an intermediate. To the extent that the chloroperoxidase-catalyzed iodination of thyroglobulin serves as a model for the thyroid coupling reaction, these results suggest that DHP is not an intermediate in thyroxine formation in the thyroid.

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