Mechanism of simultaneous iodination and coupling catalyzed by thyroid peroxidase.
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.
Taurog
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A
Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041, USA.
Dorris
M L
ML
Doerge
D R
DR
D000818:Animals; D003578:Cytochrome-c Peroxidase / Q000378:metabolism; D005609:Free Radicals; D005949:Glucose Oxidase; D006042:Goiter / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007453:Iodide Peroxidase / Q000737:chemistry / Q000378:metabolism*; D007454:Iodides / Q000378:metabolism*; D007700:Kinetics; D007784:Lactoperoxidase / Q000378:metabolism; D010544:Peroxidases / Q000378:metabolism; D012710:Serum Albumin, Bovine; D013053:Spectrophotometry; D013552:Swine; D013954:Thyroglobulin / Q000378:metabolism; D013961:Thyroid Gland / Q000201:enzymology
D005609:Free Radicals; D007454:Iodides; D012710:Serum Albumin, Bovine; D013954:Thyroglobulin; D005949:Glucose Oxidase; D006735:Horseradish Peroxidase; D007784:Lactoperoxidase; D010544:Peroxidases; C042858:lignin peroxidase; D003578:Cytochrome-c Peroxidase; D007453:Iodide Peroxidase