Anionic iodotyrosine residues are required for iodothyronine synthesis

Biosynthesis of iodothyronines in thyroglobulin occurs by oxidative coupling of two iodotyrosine residues catalyzed by thyroperoxidase. To study the mechanism of iodothyronine formation, iodine-free thyroglobulin was non-enzymatically iodinated and after removal of non-incorporated 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 thyroglobulin. T-4 is synthesized out of two di-iodotyrosine (DIT) residues in thyroglobulin. The pH dependence of T-4 formation fits the dissociation curve of the DIT phenoxy group (pK(a) 6.5). The formation of T-2, synthesized out of two mono-iodotyrosine (MIT) residues, shows a quite different pH dependence. Below pH 6, T-2 synthesis could not be observed, while above pH 7.4 a relatively large increase occurred. The Values up to pK 8 fitted the dissociation curve of the MIT-phenoxy group with a pK(a) of 8.7. The gradual loss in enzymatic activity of peroxidase and oxidase in the reaction made the values obtained above pH 8 unreliable. The importance of the ionization of the phenoxy group for the coupling reaction was further consolidated by showing that the pH-dependent oxidation of 2-methoxy-phenol (guaiacol) had 50% maximal product formation at pH 7, a Value concordant with pK(a) 7.0 for the ionization of the phenoxy group of this agent. T-3 and rT(3) Synthesis followed mainly the ionization curve of the inner-ring hydroxyl group, indicating that this ring has the greatest influence on hormonogenesis. Since anion formation facilitates the removal of an electron under oxidative conditions, the pH dependence agrees with the involvement of phenoxy radicals in iodothyronine synthesis, a process that most likely also occurs in vivo since it is mainly T-4 that is formed in thyroglobulin.