ANIONIC IODOTYROSINE RESIDUES ARE REQUIRED FOR IODOTHYRONINE SYNTHESIS

Biosynthesis of iodothyronines in thyroglobulin occurs by oxidative co upling of two iodotyrosine residues catalyzed by thyroperoxidase. To s tudy the mechanism of iodothyronine formation, iodine-free thyroglobul in was non-enzymatically iodinated and after removal of non-incorporat ed iodide, incubated with lactoperoxidase and glucose oxidase between pH 4 and 9. The amount of thyroxine (T-4), 3,5,3'-tri-iodothyronine (T -3), 3,3',5'-tri-iodothyronine (rT(3)) and 3,3'-di-iodothyronine (T-2) formed was measured by radioimmunoassays after hydrolysis of thyroglo bulin. T-4 is synthesized out of two di-iodotyrosine (DIT) residues in thyroglobulin. The pH dependence of T-4 formation fits the dissociati on curve of the DIT phenoxy group (pK(a) 6.5). The formation of T-2, s ynthesized out of two mono-iodotyrosine (MIT) residues, shows a quite different pH dependence. Below pH 6, T-2 synthesis could not be observ ed, while above pH 7.4 a relatively large increase occurred. The Value s up to pK 8 fitted the dissociation curve of the MIT-phenoxy group wi th a pK(a) of 8.7. The gradual loss in enzymatic activity of peroxidas e and oxidase in the reaction made the values obtained above pH 8 unre liable. The importance of the ionization of the phenoxy group for the coupling reaction was further consolidated by showing that the pH-depe ndent oxidation of 2-methoxy-phenol (guaiacol) had 50% maximal product formation at pH 7, a Value concordant with pK(a) 7.0 for the ionizati on of the phenoxy group of this agent. T-3 and rT(3) Synthesis followe d mainly the ionization curve of the inner-ring hydroxyl group, indica ting that this ring has the greatest influence on hormonogenesis. Sinc e anion formation facilitates the removal of an electron under oxidati ve conditions, the pH dependence agrees with the involvement of phenox y radicals in iodothyronine synthesis, a process that most likely also occurs in vivo since it is mainly T-4 that is formed in thyroglobulin .