Molecular basis for the substrate selectivity of cat type I iodothyronine deiodinase.
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 (Km) of cat D1 for rT3 (11 microm) is 30-fold higher than that of rat and human D1 (0.2-0.5 microm). Deiodination of rT3 by cat D1 is facilitated by sulfation [maximal velocity (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-Arg48-52 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.
Kuiper
George G J M
GG
Department of Internal Medicine, Room Ee 502, Erasmus Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands. g.kuiper@erasmusmc.nl.
Wassen
Frank
F
Klootwijk
Willem
W
Van Toor
Hans
H
Kaptein
Ellen
E
Visser
Theo J
TJ
D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D002415:Cats; D003001:Cloning, Molecular; D004285:Dogs; D006801:Humans; D007453:Iodide Peroxidase / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D013045:Species Specificity; D013379:Substrate Specificity; D013552:Swine; D013961:Thyroid Gland / Q000502:physiology; D014284:Triiodothyronine / Q000097:blood
D014284:Triiodothyronine; C109812:iodothyronine deiodinase type I; D007453:Iodide Peroxidase