Molecular basis for the substrate selectivity of cat type I iodothyronine deiodinase.
Molecular basis for the substrate selectivity of cat type I iodothyronine deiodinase.
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猫 I 型碘甲状腺原氨酸脱碘酶底物选择性的分子基础。
作者：
G. Kuiper;F. Wassen;W. Klootwijk;H. van Toor;E. Kaptein;T. Visser
I型碘甲状腺原氨酸脱碘酶（D1）催化T4到T3的活化以及T3 （rT3）和硫代碘甲状腺原氨酸的降解。比较了几种动物肝微粒体制剂中D1的催化活性，发现猫D1与大鼠/人D1之间存在显著差异。猫D1对rT3的米切里斯常数（Km）为11 μ m，是大鼠和人D1 （0.2 ~ 0.5 μ m）的30倍。硫酸化促进了cat D1对rT3的脱碘作用[最大速度(Vmax)/Km rT3 = 3, Vmax/Km rT3S = 81]。为了了解底物相互作用差异的分子基础，我们克隆了猫D1 cDNA，并与大鼠/人D1蛋白的氨基酸序列进行了比较。猫D1的氨基酸残基40和70之间的区域与大鼠/人D1的差异比较大。通过cat D1的定点诱变，发现需要组合突变来改善cat D1酶对rT3的脱碘作用。为了实现高效的rT3脱碘，在65位插入一个Phe，在45-46位插入Thr-Gly-Met-Thr-Arg48-52序列，以及在45-46位插入Gly和Glu氨基酸是必不可少的。这两种改变都只能有限地改善rT3脱碘。同时，所描述的突变组合并不影响已经相当有效的rT3S外环脱碘和T3S内环脱碘，而所描述的每一种变化都单独影响rT3S的脱碘。我们的研究结果表明，D1的活性位点具有很大的灵活性，可以适应各种底物。野生型猫D1的活性位点比大鼠/人D1的活性位点更不灵活，倾向于硫代碘甲状腺原氨酸。
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.
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DOI：
--
发表时间：
1994
期刊：
The Journal of biological chemistry
影响因子：
--
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DOI：
--
发表时间：
1991
期刊：
The Journal of biological chemistry
影响因子：
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作者：
Leonard,JL;Ekenbarger,DM;Frank,SJ;Farwell,AP;Koehrle,J
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DOI：
10.1210/jcem.75.4.1400883
发表时间：
1992-10
期刊：
The Journal of clinical endocrinology and metabolism
影响因子：
--
作者：
S. Mandel;M. Berry;J. Kieffer;J. Harney;R. Warne;P. Larsen
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影响因子：
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