The process of incorporation of 1 or 2 iodide atoms into the aromatic rings of a tyrosine residue of Tg followed by conversion to a thyroid hormone involves three separate steps, each of which is catalyzed by the enzyme thyroid peroxidase (TPO):
1. Oxidation of I− to Iox
2. Addition of Iox to specific tyrosine acceptor residues of thyroglobulin, particularly at the hormonogenic and donor sites (Figure 1)
3. Coupling of two iodotyrosyl residues to generate one thyroid hormone residue and a corresponding dehydroalanine residue within the thyroglobulin molecule.
Fig1. Hormonogenic sites of thyroglobulin. The peptide backbone of thyroglobulin is represented by the black line. Certain tyrosine residues are specified by their position from the N-terminal. The three main sites at which T4 is found are shown by blue circles; the orange square is the site at which T3 is usually found. These are acceptor sites during the coupling reaction. Purple triangles show the positions of other iodinated tyrosyl residues; these are likely donor sites from which the outer ring of T3 or T4 is derived during the coupling reaction. B. The amino acid sequences surrounding the four hormonogenic sites in A are given.
As with Tg, the enzyme TPO, a heme-containing 933 amino acid member of the peroxidase family, is synthesized on the rough endoplasmic reticulum of the thyrocyte. It is processed, including glycosylation, by the Golgi apparatus and is ultimately anchored in the apical membrane by a hydrophobic region in its C-terminal region. Most of the protein, including its catalytic domain, is located in the lumen of the follicle.
Figure 2 shows TPO in the apical membrane of the thyrocyte, along with the enzyme responsible for generating the H2O2 required for the peroxidase activity of TPO. This Ca2+/NADPH-dependent oxidase, which is expressed abundantly but not exclusively in the thyroid gland, is called dual oxidase, DUOX, because it contains both an NADPH oxidase domain and peroxidase activity. When there is sufficient I− available, H2O2 production by DUOX is the rate-limiting step in thyroid hormone production. High levels of iodine inhibit the enzyme’s activity and TSH stimulates it. There are two forms of the enzyme, DUOX1 and DUOX2, which are encoded on separate genes on chromosome 20. Both DUOX1 and DUOX2 require specific maturation factors, DUOXA1 and DUOXA2, to ensure their proper location and activity in the cell. The genes for these maturation proteins are located in the intergenic region between the the genes for the two enzymes. In addition to the thyroid gland, these enzymes appear in other tissues to support varying ion transport functions. Mutations in DUOX2 or DUOXA2 may result in congenital hypothyroidism, but the range of phenotypes observed suggest that the extent of condition may be influenced by other factors.
Fig2. Thyroid hormone synthesis by thyroid peroxidase (TPO). In this schematic diagram, two tyrosine residues are represented by the blue (hormonogenic site) and purple (donor site) circles. In the reactions catalyzed by TPO, H2O2 generated by the dual oxidase, DUOX, is required for both the iodination of these residues and for their coupling to form T4 (see Figure 3). The residue remaining at the latter site after the coupling is dehydroalanine, represented by the purple square.
In the first step catalyzed by TPO, the H2O2 generated by DUOX is used to oxidize I−, as depicted in Figure 2. This oxidized I− (Iox), the exact nature of which has not yet been definitively elucidated, is incorporated into a tyrosine ring of thyroglobulin to form either a monoiodotyrosine (MIT) or diiodotyrosine (DIT) residue of the protein. One of the major physiological controls of the abundance of iodotyrosine, iodothyronine, and therefore thyroid hormone in thyroglobulin, is the dietary iodide supply. In the absence of ample dietary iodine the amount of thyroid hormone produced per molecule of thyroglobulin falls due to its sparse iodination. Iodination of thyroglobulin is also inhibited by excess iodide; this is known as the Wolff Chaikoff effect, a transient phenomenon to which the thyrocyte adapts by “escaping” the inhibition. Thus, proper functioning of this stage of thyroid hormone syn thesis, iodination of tyrosine residues, is dependent on adequate but not excessive levels of iodine availability.
Finally, TPO catalyzes the coupling between two iodinated tyrosine residues, also depicted in Figure 2 and in more detail in Figure 3. In this reaction, an iodinated (a diiodinated ring is depicted in Figure 1) tyrosyl ring is removed from a donor site (see Figure 1) on thyroglobulin and becomes the outer ring of T4 at the hormonogenic site. This leaves behind dehydroalanine at the donor site. If the tyrosine at the donor site had been monoiodinated, the resulting molecule at the hormonogenic site would be T3. A typical thyroglobulin molecule from an individual with normal thy roid status contains about 5 residues of each MIT and DIT, 2.5 residues of T4, and 0.7 residues of T3.
Fig3. TPO-catalyzed coupling of iodotyrosine residues. The outer ring of T4 or T3 being formed at the hormonogenic site is derived from a donor site. It has not yet been elucidated whether the intermediate is a free radical or is ionic.
