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Molecular Basis for the Substrate Selectivity of Cat Type I Iodothyronine Deiodinase

Department of Internal Medicine, Erasmus Medical Center, 3000 DR Rotterdam, The Netherlands

Address all correspondence and requests for reprints to: George Kuiper, Ph.D., Department of Internal Medicine, Room Ee 502, Erasmus Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands. E-mail: g.kuiper{at}erasmusmc.nl.

The type I iodothyronine deiodinase (D1) catalyzes the activation of T₄ to T₃ as well as the degradation of T₃ (rT₃) and sulfated iodothyronines. A comparison of the catalytic activities of D1 in liver microsomal preparations from several species revealed a remarkable difference between cat D1 on one hand and rat/human D1 on the other hand. The Michaelis constant (K_m) of cat D1 for rT₃ (11 µM) is 30-fold higher than that of rat and human D1 (0.2–0.5 µM). Deiodination of rT₃ by cat D1 is facilitated by sulfation [maximal velocity (V_max)/K_m rT₃ = 3 and V_max/K_m rT₃S = 81]. To understand the molecular basis for the difference in substrate interaction the cat D1 cDNA was cloned, and the deduced amino acid sequence was compared with rat/human D1 protein. In the region between amino acid residues 40 and 70 of cat D1, various differences with rat/human D1 are concentrated. By site-directed mutagenesis of cat D1 it was found that a combination of mutations was necessary to improve the deiodination of rT₃ by cat D1 enzyme. For efficient rT₃ deiodination, a Phe at position 65 and the insertion of the Thr-Gly-Met-Thr-Arg^48–52 sequence as well as the amino acids Gly and Glu at position 45–46 are essential. Either of these changes alone resulted in only a limited improvement of rT₃ deiodination. At the same time the combination of the described mutations did not affect the already quite efficient outer ring deiodination of rT₃S nor the inner ring deiodination of T₃S, whereas each of the described changes alone did affect rT₃S deiodination. Our findings suggest great flexibility of the active site in D1 that adapts to its various substrates. The active site of wild-type cat D1 is less flexible than the active site of rat/human D1 and favors sulfated iodothyronines.

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