Mechanism of simultaneous iodination and coupling catalyzed by thyroid peroxidase
Mechanism of simultaneous iodination and coupling catalyzed by thyroid peroxidase
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DOI：
10.1006/abbi.1996.0222
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发表时间：
1996-06-01
影响因子：
3.9
通讯作者：
Doerge, DR
中科院分区：
文献类型：
--
作者：
Taurog, A;Dorris, ML;Doerge, DR
甲状腺过氧化物酶（TPO）在甲状腺中同时催化两种不同类型的反应——碘化和偶联，本研究探讨了这种同时双重活性的机制，TPO的双电子氧化产物化合物I以两种不同的形式存在-氧铁基卟啉-阳离子自由基和氧铁基蛋白自由基，提出了碘化是由TPO化合物I的卟啉-阳离子自由基形式介导的。然而，本研究的结果支持化合物I的卟啉-阳离子自由基形式介导碘化和偶联的观点。在研究的第一部分，我们比较了两种晶体结构非常相似的过氧化物酶-细胞色素c过氧化物酶（CcP）和木质素过氧化物酶（LiP）的偶联和碘化活性，尽管这两种过氧化物酶具有非常相似的三维结构。两种酶的催化活性比较表明，二碘酪氨酸（diiodotyrosine， DIT）刺激下的LiP偶联活性显著大于CcP。此外，木质素过氧化物酶在酸性ph下表现出非常显著的碘化活性，而CcP在所有ph下的碘化作用都可以忽略不计。我们对这两种结构相似的过氧化物酶的研究结果表明，TPO催化的碘化和偶联都可能是由化合物I的卟啉-阳离子自由基形式介导的，本研究的第二部分采用TPO和乳酸过氧化物酶（LPO）模型系统，在该模型系统中碘化和偶联同时发生，获得了更直接的证据支持这一观点。利用血红素光谱分析将化合物I蛋白自由基形式的形成与碘化和偶联反应动力学联系起来，直到碘化和偶联反应几乎完成时才观察到化合物I蛋白自由基的形成。在单独的实验中，TPO或LPO化合物I的卟啉-阳离子自由基形式向蛋白质自由基形式的自发转化被低浓度的碘明显抑制，特别是在碘受体存在的情况下。这些研究提供了令人注目的证据，证明碘化和偶联都是由化合物I的卟啉-阳离子自由基形式介导的。这一发现进一步证实了偶联在过量碘的存在下被抑制，这一观察结果很容易解释为碘和DIT残基在甲状腺球蛋白中被卟啉-阳离子自由基氧化的竞争。(C) 1996学术出版社，Inc.
Thyroid peroxidase (TPO) simultaneously catalyzes two very different types of reaction in the thyroid gland-iodination and coupling, The present study addresses the mechanism of this simultaneous dual activity, Compound I, the two-electron oxidation product of TPO, exists in two different forms-an oxoferryl porphyrin pi-cation radical and an oxoferryl protein radical, It has been proposed that iodination is mediated by the porphyrin pi-cation radical form of TPO compound I, while coupling is mediated by the protein radical form, However, results obtained in the present study favor the view that both iodination and coupling are mediated by the porphyrin pi-cation radical form of compound I, In the first part of the study, we compared coupling and iodination activities of two peroxidases with very similar crystal structures-cytochrome c peroxidase (CcP) and lignin peroxidase (LiP), Although these two peroxidases have very similar three-dimensional structures, CcP forms a compound I only of the protein radical type, whereas compound I of LiP exists only as a porphyrin pi-cation radical, Comparison of the catalytic activities of the two enzymes showed that diiodotyrosine (DIT)-stimulated coupling activity of LiP was significantly greater than that of CcP, Moreover, lignin peroxidase displayed very significant iodinating activity at acid pHs, whereas iodination with CcP was negligible at all pHs tested, Our findings with these two structurally similar peroxidases suggested that TPO-catalyzed iodination and coupling could both be mediated by the porphyrin pi-cation radical form of compound I, More direct evidence in support of this view was obtained in the second part of this study, employing TPO and lactoperoxidase (LPO) model systems in which iodination and coupling occurred simultaneously, Heme spectral analysis was used to correlate formation of the protein radical form of compound I with the kinetics of the iodination and coupling reactions, Formation of the compound I protein radical was not observed until the iodination and coupling reactions had almost been completed, In separate experiments it was shown that the spontaneous conversion of the porphyrin pi-cation radical form of TPO or LPO compound I to the protein radical form was markedly inhibited by a low concentration of iodide, especially in the presence of an iodide acceptor, These studies provide compelling evidence that both iodination and coupling are mediated by the porphyrin pi-cation radical form of compound I, This was further substantiated by the finding that coupling was inhibited in the presence of excess iodide, an observation readily explained by competition between iodide and DIT residues in thyroglobulin for oxidation by the porphyrin pi-cation radical. (C) 1996 Academic Press, Inc.