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The Role of the Active Site Cysteine in Catalysis by Type 1 Iodothyronine Deiodinase¹

Thyroid Division, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: P. Reed Larsen, M.D., Thyroid Division, Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115. E-mail: larsen{at}rascal.med.harvard.edu

Type 1 iodothyronine deiodinase (deiodinase 1) is a selenoenzyme that converts the prohormone T₄ to the active thyroid hormone T₃ by outer ring deiodination or to the inactive metabolite rT₃ by inner ring deiodination. Although selenocysteine has been demonstrated to be essential for the biochemical profile of deiodinase 1, the role of a highly conserved, active site cysteine (C124 in rat deiodinase 1) has not been defined. The present studies examined the effects of a Cys¹²⁴Ala mutation on rat deiodinase 1 enzymatic function and substrate affinity. At a constant 10-mM concentration of dithiothreitol (DTT), the C124A mutant demonstrated a 2-fold lower apparent maximal velocity (V_max) and K_m for rT₃ (K_mrT₃) than the wild type for outer ring deiodination, whereas the V_max/K_m ratio was unchanged. Similarly, the apparent V_max and K_mT₃ sulfate for inner ring deiodination were 2-fold lower in the C124A mutant relative to those in the wild type, with no change in the V_max/K_m ratio. The C124A mutant exhibited ping-pong kinetics in the presence of DTT, and substitution of the active site cysteine increased the K_mDTT by 14-fold relative to that of the wild-type enzyme, with no significant effects on K_mrT₃ or V_max. The C124A mutant was inhibited by propylthiouracil in an uncompetitive fashion and exhibited a 2-fold increase in K_ipropylthiouracil compared with that of the wild type. K_mrT₃ was also reduced for the C124A mutant when 5 mM reduced glutathione, a potential physiological monothiol cosubstrate, was used in outer ring deiodination assays. These results demonstrate that thiol cosubstrate interactions with C124 in type 1 deiodinase play an important role in enhancing catalytic efficiency for both outer and inner ring deiodination.

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