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This version published online on September 4, 2003
Endocrinology, doi:10.1210/en.2003-0728
A more recent version of this article appeared on December 1, 2003
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Submitted on June 10, 2003
Accepted on August 6, 2003

MOLECULAR BASIS FOR THE SUBSTRATE SELECTIVITY OF CAT TYPE I IODOTHYRONINE DEIODINASE

George G. J. M. Kuiper1*, Frank Wassen1, Willem Klootwijk1, Hans van Toor1, Ellen Kaptein1, and Theo J. Visser1

1 Department of Internal Medicine, Erasmus Medical Center, PO Box 1738, 3000 DR Rotterdam, The Netherlands

* To whom correspondence should be addressed. E-mail: g.kuiper{at}erasmusmc.nl.

The type I iodothyronine deiodinase (D1) catalyzes the activation of thyroxine (T4) to 3,5,3'-triiodothyronine (T3) as well as the degradation of 3,3',5'-triiodothyronine (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 Km 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 (Vmax/Km rT3 = 3 and Vmax/Km 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, while each of the described changes alone did affect rT3S deiodination. Our findings suggest great flexibility of the active site in D1 which 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.
Key words: iodothyronines • deiodination • type I deiodinase • thyroxine • sulfated iodothyronines




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