Yamamoto T, Nozaki A, Shintani S, Ishikura S, Katagiri Y, Hara A
Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 502-8585, Japan.
J Biochem. 2000 Jul;128(1):121-8. doi: 10.1093/oxfordjournals.jbchem.a022722.
The NADP(H)-linked oxidoreductase activity of a major isozyme of human liver 3 alpha-hydroxysteroid dehydrogenase was activated 5-, 4-, and 2-fold by D-thyroxine (T(4)), L-T(4) and DL-3,3', 5'-triiodothyronine (reverse T(3)), respectively. Kinetic analysis of the activation indicated that D-T(4), L-T(4), and reverse T(3) are non-essential activators, showing binding constants of 1.5, 1.1, and 3.6 microM, respectively. Comparison of the effects of the T(4) analogs on the activities of the mutant enzymes suggests that the binding site is composed of at least Lys-270, Arg-276, and the C-terminal loop of the enzyme. L-T(3), DL-thyronine, and D-tyrosine had no effect on the enzyme, but 3,5,3',5'-tetra- and 3,5, 3'-tri-iodo thyropropionic acids were potent competitive inhibitors with K(i) values of 42 and 60 nM, respectively, with respect to the substrate. The inhibition constant was lowered upon the activation of the enzyme by D-T(4), and the inhibition by the deamino derivatives of T(4) and T(3) disappeared upon modification of the C-terminal loop of the enzyme, but not upon replacement of Lys-270 or Arg-276 with Met. These results indicate that, depending on their structures, the T(4) analogs bind differently to two distinct sites at the active center of the enzyme to produce stimulatory and inhibitory effects.
人肝脏3α-羟基类固醇脱氢酶一种主要同工酶的NADP（H）连接氧化还原酶活性分别被D-甲状腺素（T4）、L-T4和DL-3,3',5'-三碘甲状腺原氨酸（反式T3）激活了5倍、4倍和2倍。对这种激活作用的动力学分析表明，D-T4、L-T4和反式T3是非必需激活剂，其结合常数分别为1.5、1.1和3.6微摩尔。比较T4类似物对突变酶活性的影响表明，结合位点至少由酶的赖氨酸-270、精氨酸-276和C末端环组成。L-T3、DL-甲状腺素和D-酪氨酸对该酶无影响，但3,5,3',5'-四碘和3,5,3'-三碘甲状腺丙酸是有效的竞争性抑制剂，相对于底物的抑制常数（Ki）值分别为42和60纳摩尔。当酶被D-T4激活时，抑制常数降低，并且当酶的C末端环被修饰时，T4和T3的脱氨基衍生物的抑制作用消失，但用甲硫氨酸取代赖氨酸-270或精氨酸-276时抑制作用不消失。这些结果表明，根据其结构，T4类似物以不同方式结合到酶活性中心的两个不同位点，从而产生刺激和抑制作用。
