Substitution of cysteine for selenocysteine in the catalytic center of type III iodothyronine deiodinase reduces catalytic efficiency and alters substrate preference.
Human type III iodothyronine deiodinase (D3) catalyzes the conversion of T(4) to rT(3) and of T(3) to 3, 3'-diiodothyronine (T2) by inner-ring deiodination. Like types I and II iodothyronine deiodinases, D3 protein contains selenocysteine (SeC) in the highly conserved core catalytic center at amino acid position 144. To evaluate the contribution of SeC144 to the catalytic properties of D3 enzyme, we generated mutants in which cysteine (D3Cys) or alanine (D3Ala) replaces SeC144 (D3wt). COS cells were transfected with expression vectors encoding D3wt, D3Cys, or D3Ala protein. Kinetic analysis was performed on homogenates with dithiothreitol as reducing cofactor. The Michaelis constant of T(3) was 5-fold higher for D3Cys than for D3wt protein. In contrast, the Michaelis constant of T(4) increased 100-fold. The D3Ala protein was enzymatically inactive. Semiquantitative immunoblotting of homogenates with a D3 antiserum revealed that about 50-fold higher amounts of D3Cys and D3Ala protein are expressed relative to D3wt protein. The relative substrate turnover number of D3Cys is 2-fold reduced for T(3) and 6-fold reduced for T(4) deiodination, compared with D3wt enzyme. Studies in intact COS cells expressing D3wt or D3Cys showed that the D3Cys enzyme is also active under in situ conditions. In conclusion, the SeC residue in the catalytic center of D3 is essential for efficient inner-ring deiodination of T(3) and in particular T(4) at physiological substrate concentrations.
Department of Internal Medicine, Erasmus Medical Center, 3000 DR Rotterdam, The Netherlands. g.kuiper@erasmusmc.nl
George G J M
GG
Kuiper
Willem
W
Klootwijk
Theo J
TJ
Visser
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain / Q000235:genetics*; D003545:Cysteine / Q000235:genetics; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D007453:Iodide Peroxidase / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017279:Selenocysteine / Q000235:genetics; D013379:Substrate Specificity; D013974:Thyroxine / Q000378:metabolism; D014284:Triiodothyronine / Q000378:metabolism
D014284:Triiodothyronine; D017279:Selenocysteine; C117274:iodothyronine deiodinase type III; D007453:Iodide Peroxidase; D003545:Cysteine; D000409:Alanine; D013974:Thyroxine