Kuiper George G J M, Klootwijk Willem, Visser Theo J
Department of Internal Medicine, Erasmus University Medical Center, 3000 DR Rotterdam, The Netherlands.
Endocrinology. 2002 Apr;143(4):1190-8. doi: 10.1210/endo.143.4.8738.
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.
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.
人II型碘甲状腺原氨酸脱碘酶（D2）催化T4转化为T3。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一个硒代半胱氨酸（SeC）残基（D2中的第133位残基）。值得注意的是，迄今为止克隆的所有D2蛋白在SeC的两个残基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与辅因子或酶残基参与氧化还原反应来协助酶促作用。我们研究了在SeC的两个残基氨基末端具有半胱氨酸（A131C）或丝氨酸（A131S）的D2突变体是否具有酶活性，并对这些突变体的底物亲和力、还原辅因子相互作用和抑制剂谱进行了表征。用编码野生型（wt）D2、D2 A131C或D2 A131S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 A131C和A131S突变体对T4（5 nM）和反T3（9 nM）的米氏常数与野生型D2酶相似。D2 A131C酶对DTT的极限米氏常数比野生型D2酶低3倍。在20 mM DTT存在下，野生型和突变型D2酶对丙硫氧嘧啶基本不敏感[抑制50%活性的浓度（IC50）>2 mM]，但在0.2 mM DTT存在下测试时，丙硫氧嘧啶的IC50值降至约0.1 mM。在表达野生型D2、D2 A131C或D2 A131S的完整COS细胞孵育过程中，添加越来越多的未标记T4导致[125I]T4脱碘饱和，这反映在释放到培养基中的[125I]T3减少。饱和首先出现在培养基T4浓度在1至10 nM之间。
在D2蛋白的催化中心将保守的丙氨酸残基替换为半胱氨酸在体外和原位均不会使酶失活，反而在体外改善了与还原辅因子DTT的相互作用。
