Substitution of cysteine for a conserved alanine residue in the catalytic center of type II iodothyronine deiodinase alters interaction with reducing cofactor.
Substitution of cysteine for a conserved alanine residue in the catalytic center of type II iodothyronine deiodinase alters interaction with reducing cofactor.
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DOI：
10.1210/endo.143.4.8738
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发表时间：
2002-04
期刊：
影响因子：
4.8
通讯作者：
G. Kuiper;W. Klootwijk;T. Visser
中科院分区：
文献类型：
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作者：
G. Kuiper;W. Klootwijk;T. Visser
未标记人II型碘甲状腺原氨酸脱碘酶（D2）催化T（4）活化为T（3）。与I型（D1）和III型（D3）脱碘酶一样，D2酶在高度保守的催化中心含有硒代半胱氨酸（SeC）残基（D2中的残基133）。值得注意的是，迄今为止克隆的所有D2蛋白在SeC的氨基末端都有一个丙氨酸，而所有D1和D3蛋白在这个位置都含有一个半胱氨酸。催化中心的半胱氨酸残基可以通过提供亲核硫化物或通过参与与辅因子或酶残基的氧化还原反应来帮助酶促作用。我们已经调查了是否D2突变体的半胱氨酸（A131 C）或丝氨酸（A131 S）的两个残基的氨基末端的SeC是酶活性和特征在于这些突变体的底物亲和力，减少辅因子的相互作用和抑制剂的配置文件。用编码野生型（wt）D2、D2 A131 C或D2 A131 S蛋白的表达载体转染COS细胞。以二硫苏糖醇（DTT）作为还原辅因子，对匀浆进行动力学分析。D2 A131 C和A131 S突变体显示出与野生型D2酶相似的T（4）（5 nM）和反向T（3）（9 nM）的Michaelis-Menten常数值。D2 A131 C酶的DTT的极限米氏常数比野生型D2酶低3倍。在20 mM DTT存在下，wt和突变体D2酶基本上对丙基硫氧嘧啶不敏感[抑制50%活性的浓度（IC（50））> 2 mM]，但当在0.2 mM DTT存在下测试时，丙基硫氧嘧啶的IC（50）值降低至约0.1 mM。或D2 A131 S中，增加未标记T（4）的量导致[（125）I]T（4）脱碘饱和，这反映在[（125）I]T（3）释放到培养基中的减少。饱和首先出现在1和10 nM之间的中等T（4）浓度。结论半胱氨酸取代D2蛋白催化中心的保守丙氨酸残基不会在体外和原位破坏酶，而是改善了与还原辅因子DTT的体外相互作用。
UNLABELLED Human type II iodothyronine deiodinase (D2) catalyzes the activation of T(4) to T(3). The D2 enzyme, like the type I (D1) and type III (D3) deiodinases, contains a selenocysteine (SeC) residue (residue 133 in D2) in the highly conserved catalytic center. Remarkably, all of the D2 proteins cloned so far have an alanine two residue-amino terminal to the SeC, whereas all D1 and D3 proteins contain a cysteine at this position. A cysteine residue in the catalytic center could assist in enzymatic action by providing a nucleophilic sulfide or by participating in redox reactions with a cofactor or enzyme residues. We have investigated whether D2 mutants with a cysteine (A131C) or serine (A131S) two-residue amino terminal to the SeC are enzymatically active and have characterized these mutants with regard to substrate affinity, reducing cofactor interaction and inhibitor profile. COS cells were transfected with expression vectors encoding wild-type (wt) D2, D2 A131C, or D2 A131S proteins. Kinetic analysis was performed on homogenates with dithiothreitol (DTT) as reducing cofactor. The D2 A131C and A131S mutants displayed similar Michaelis-Menten constant values for T(4) (5 nM) and reverse T(3) (9 nM) as the wt D2 enzyme. The limiting Michaelis-Menten constant for DTT of the D2 A131C enzyme was 3-fold lower than that of the wt D2 enzyme. The wt and mutant D2 enzymes are essentially insensitive to propylthiouracil [concentration inhibiting 50% of activity (IC(50)) > 2 mM] in the presence of 20 mM DTT, but when tested in the presence of 0.2 mM DTT the IC(50) value for propylthiouracil is reduced to about 0.1 mM. During incubations of intact COS cells expressing wt D2, D2 A131C, or D2 A131S, addition of increasing amounts of unlabeled T(4) resulted in the saturation of [(125)I]T(4) deiodination, as reflected in a decrease of [(125)I]T(3) release into the medium. Saturation first appeared at medium T(4) concentrations between 1 and 10 nM. IN CONCLUSION substitution of cysteine for a conserved alanine residue in the catalytic center of the D2 protein does not inactivate the enzyme in vitro and in situ, but rather improves the interaction with the reducing cofactor DTT in vitro.