Endocrine Journal 1996, 43 (1), 1-14 Review Thyroid Peroxidase: Experimental and Clinical Integration SACHIYAOHTAKI, HIDEHIKO NAKAGAWA, MASAO NAKAMURA*, AND ToMlo KOTANI Department of Laboratory Medicine, Miyazaki Medical College, Miyazaki 889-16, and *Biophysics, Research Institute for Electronic Science, Hokkaido University, Sapporo 060, Japan tyrosine residues and the oxidative coupling of two Introduction iodotyrosine residues on thyroglobulin (Tg) [1-4]. TPO requires both iodide and H2O2 to initiate This article aims to review the recent advances hormone synthesis. Iodine is a trace element that in knowledge concerning thyroid peroxidase (TPO), is incorporated as inorganic iodide across the thy- focusing on experimental and clinical integration. roid cell membrane through the Na+-I--cotransport This is a propitious time to examine the topic, mechanism [5-7]. The NADPH-dependent O2 gen- because recent advances in the mechanisms of thy- erating system is isolated from the thyroid plasma roid hormone biosynthesis and increased membrane, where thyroid hormone synthesis main- understanding of the molecular biology of TPO ly occurs [8-10]. The resulting superoxide anions have provided a basis for obtaining much impor- dismutate rapidly to H2O2 through the action of tant knowledge of various thyroid diseases such superoxide dismutase, which is present in cytosol as autoimmune thyroiditis. and cell fragments. Pommier and coworkers [11, These developments, which we discuss and em- 12] have isolated a different H202-generating sys- phasize, are: first of all, mechanisms of thyroid tem, which produces H2O2 directly at the expense hormone synthesis catalyzed by this enzyme, in- of NADPH. cluding its structure, enzyme activity, and In addition to the stimulation of iodide trans- regulation of synthesis; secondly, gene structure port and H2O2 generation in the thyroid, the protein and transcription mechanisms; thirdly, mutation synthesis of Tg and TPO in response to TSH has of the TPO gene in hitherto reported cases, and been reported [3, 5]. The reaction mechanism of finally, the critical implications of TPO as an au- TPO and the regulation of thyroid hormone syn- toantigen causing experimental focal thyroiditis. thesis in the TPO reaction will be described in the next section. I. Thyroid Hormone Synthesis B. Properties of TPO Catalyzed by TPO TPO, which is a membrane-bound hemoprotein, A. Iodide transport and H2O2 generation has been purified from thyroid microsomes by pro- cedures involving trypsin digestion and detergent TPO is involved in two different reactions in the treatment [13-17]. The enzyme thus purified has biosynthesis of thyroid hormone: the iodination of been used for studies of its mechanism of reaction. By monoclonal antibody-assisted immunoaffinity This review was written as a memorial article for the chromatography, Nakagawa et al. [16] have ob- Miyake Prize of Japan, which was awarded to one of the tained a native-type enzyme from hog thyroid authors, Dr. S. Ohtaki, on October 20, 1994. Correspondence to: Dr. Sachiya OHTAKI, Department of microsomes with a molecular weight of 100 kDa. Laboratory Medicine, Miyazaki Medical College, 5200 The molecular weight of the enzyme is similar to Kihara, Kiyotakecho, Miyazaki 889-16, Japan that deduced from cDNA [18, 19], and no differ- 2 OHTAKI et al. ence is observed in the specific activities estimated from the ratio of activities to the heme concentra- tion of TPO [20]. The visible and pyridine hemochrome spectra of TPO are very similar to those of lactoperoxidase (LPO) [17]. The heme prosthetic group of LPO is concluded to be an iron protoporphyrin thiol which is bound to the cysteine residue of the apoprotein via a disulfide bridge [21]. The similarities in nu- cleotide and amino acid sequences between TPO and myeloperoxidase (MPO) in animal peroxidase have been presented, and candidates for the fifth and sixth iron ligand histidines in these heme per- oxidases have been suggested [19]. Recently, the same similarity of LPO to TPO and MPO and the positions of half cystine in the amino acid sequence of LPO binding to heme thiol have been suggested by Cals et al. [22]. It is not surprising, therefore, that TPO and LPO catalyze both iodinating and coupling reactions. C. Iodinating activity of TPO Scheme 1. Mechanism of reactions catalyzed by thyroid Peroxidase reactions catalyzed by heme peroxi- peroxidase. Values in parenthesis denote dases can be studied with horseradish peroxidase second order rate constants (M-'s-'). Path 1 and Path 2 indicate the coupling and iodinating (HRP) and LPO. Inorganic iodide in the thyroid cycles, respectively. Tyr, tyrosine; MIT, monoiodotyrosine; DIT, diiodotyrosine; 0.2% I 7.5 x 106M-'s-' TPO + H2O2 >Compound I ()1 Tg, 0.2% iodine-content thyroglobulin. 1-5 x 104M-'s~' C ompound I + Tyrosine - TPO + Tyrosine (2) radical reactions: iodotyrosine is formed at the ac- tive site of TPO through the reaction of iodide 2 x 10'M-'s-' C ompound I + I- TPO•I+ (3) radicals with tyrosine radicals. Conclusive evi- dence that TPO catalyzes two-electron oxidation 2.1 x 106M-'s-' TPO •I+ + Tyrosine 'TPO + MIT (4) of tyrosine has been reported [20, 23, 26] (equation 2). One can therefore rule out the possibility that gland must be first oxidized to a higher oxidation the iodinating cycle is a radical reaction. Bjorksten form as an iodinating agent. TPO is a catalyst [27] has concluded that HRP Compound I is re- which oxidizes iodide in the presence of H202. The duced directly to the ferric form with iodide. The reactions of TPO with H2O2 and iodide are very two-electron oxidation of iodide by LPO and TPO, similar to those reported with LPO [20, 23]. Rest- resulting in the formation of iodinium cation, has ing TPO (ferric) reacts with H2O2 to form a catalytic been confirmed in later studies [20, 23] (equation intermediate referred to as Compound I, which has 3). The rate of reaction of TPO Compound I with two oxidizing equivalents above the ferric form iodide is 100 fold higher than that with tyrosine (equation 1). The hypervalent enzyme mostly cat- [28]. Iodinating reaction (Path 2) therefore pro- alyzes consecutive one-electron oxidation of organic ceeds predominantly even in the presence of a low substrates through Compound II, forming substrate concentration of iodide [20, 28]. The iodinating radicals. The mechanism of the peroxidase reac- intermediate, TPO•I+, is assumed to be present from tion has been shown as Path 1 (Scheme 1). Early the overall kinetics [28]. By kinetically analyzing reports [1, 24, 25] have shown that iodinating reac- the iodinating reaction of tyrosine catalyzed by tions catalyzed by peroxidases are presented by TPO, the rate constant for the reaction of the io- REVIEW: THYROID PEROXIDASE 3 dinium cation transfer from the enzyme to tyrosine photometric analysis of the catalytic intermediates is calculated to be 2.1 x 106 M-1s-1 [28] (equation 4). of enzyme in the oxidation of various phenols, it These results indicate that the rate-determining step can be concluded that the mechanism of oxidation in the iodinating reaction is the iodinium cation catalyzed by TPO changes from a two-electron to transfer to tyrosine (Path 2). The rate constants for a one-electron type as the substituents at the l- the iodinium cation transfer to Tg have also been and 6-positions of phenol become bulky or heavy determined from overall kinetic experiments on the [20]. iodination of Tg [29]. Questions will arise as to whether the same The rate constants decrease as the iodine con- mechanism can be applied to the oxidation of Tgs tent of Tg increases. This iodine-transferring that have different iodine contents. The enzyme reaction is increased in the presence of iodothy- intermediate in the steady state of oxidation of Tg ronines in the order of diiodothyronine (T2) containing 0.2% iodine is Compound I, whereas >triiodothyronine (T3)>thyroxine (T4), similarly to Compound II is observed in the oxidation of Tg the reaction of tyrosine [28]. containing 0.7% iodine [32]. The number of DIT The mechanism of action of thioureylene anti- residues in Tg changes from 1.1 to 8.7 as the io- thyroid compounds has been examined by many dine content increases from 0.2 to 0.7% [33]. The workers [1, 3], and has been explained on the basis results indicate that TPO catalyzes two-electron of the iodinating cycle of TPO. Methylmercap- oxidation of Tg with 0.2% iodine and one-electron toimidazole (MMI), a typical goitrogen, inhibits the oxidation of 0.7% iodine-containing Tg (Scheme 1). iodinating activity of TPO in two ways. First, MMI ESR detection of 0.7% iodine-containing Tg radi- competes with Tg for the reaction with TPO•I+ [30, cals is unsuccessful, but 0.7% iodine-containing 31]. The spectrum of iodinated MMI is optically Tg-mediated ascorbate radical and GSH radical for- discernible with that of MMI. Second, MMI reacts mation catalyzed by TPO is observed [32]. rapidly with Compound I to form Compound II, Phenoxyl radicals are unstable but are readily scav- which subsequently reacts with MMI, forming the enged by ascorbate and GSH, leading to the sulf form [30]. The formation of either Compound corresponding radicals [34]. These results indicate II or the sulf form causes TPO to deviate from the that DIT residues in 0.7% iodine-containing Tg are iodinating cycle (Path 2). oxidized mainly to radicals and imply that the ox- idative coupling cycle is a radical reaction (Path D. Oxidative coupling by TPO 1). Neither significant oxidation of Tg nor 0.7% iodine-containing Tg-mediated radical formation by LPO is observed [32]. Thus it seems that, un- > 105M-1 s 1 C ompound I + DIT -- - like iodination, the oxidation of Tg occurs through -~ Compound 11+ DIT • (5) a more specific interaction with TPO.