Goswami A, Rosenberg I N
J Clin Invest. 1984 Dec;74(6):2097-106. doi: 10.1172/JCI111634.
The thiol-activated enzymatic outer-ring monodeiodination of iodothyronines by rat kidney microsomes at low (nanomolar) substrate concentrations shows an apparently sequential reaction mechanism and is further characterized by insensitivity to inhibition by dicoumarol, a moderate sensitivity to inhibition by propylthiouracil (Ki = 100 microM) and iopanoic acid (Ki = 0.9 mM), responsiveness to 5 mM glutathione (GSH), and a thermal activation profile that is concave downward with a Td of approximately 20 degrees C. In contrast, the activity at high (micromolar) substrate concentrations shows a ping-pong reaction mechanism, is inhibited by micromolar concentrations of propylthiouracil, iopanoic acid and dicoumarol, is unresponsive to 5 mM GSH, and shows a concave upward thermal activation profile. Analysis of the microsomal deiodinase reaction over a wide range of 3,3',5'-triiodothyronine (rT3) concentrations (0.1 nM to 10 microM) suggested the presence of two enzymatic activities, with apparent Michaelis constants (Km) of 0.5 microM and 2.5 nM. Lineweaver-Burk plots of reaction velocities at nanomolar substrate concentrations in presence of 100 microM propylthiouracil also revealed an operationally distinct enzymatic activity with Km's of 2.5 and 0.63 nM and maximum velocities (Vmax's) of 16 and 0.58 pmol/mg protein per h for rT3 and thyroxine (T4), respectively. These findings are consistent with the presence of a low Km iodothyronine 5'-deiodinase in rat kidney microsomes distinct from the well characterized high Km enzyme and suggest that at circulating levels of free T4 the postulated low Km enzyme could be physiologically important.
在低（纳摩尔）底物浓度下，大鼠肾微粒体对碘甲状腺原氨酸进行硫醇激活的酶促外环单脱碘反应，呈现出明显的顺序反应机制，其进一步特征包括对双香豆素抑制不敏感、对丙硫氧嘧啶（Ki = 100 μM）和碘番酸（Ki = 0.9 mM）抑制具有中等敏感性、对5 mM谷胱甘肽（GSH）有反应，以及热激活曲线向下凹，Td约为20℃。相比之下，在高（微摩尔）底物浓度下的活性呈现乒乓反应机制，受到微摩尔浓度的丙硫氧嘧啶、碘番酸和双香豆素抑制，对5 mM GSH无反应，且热激活曲线向上凹。在广泛的3,3',5'-三碘甲状腺原氨酸（rT3）浓度范围（0.1 nM至10 μM）内对微粒体脱碘酶反应进行分析，提示存在两种酶活性，其表观米氏常数（Km）分别为0.5 μM和2.5 nM。在100 μM丙硫氧嘧啶存在下，对纳摩尔底物浓度时反应速度的Lineweaver-Burk作图也显示出一种操作上不同的酶活性，rT3和甲状腺素（T4）的Km分别为2.5和0.63 nM，最大速度（Vmax）分别为每小时每毫克蛋白16和0.58 pmol。这些发现与大鼠肾微粒体中存在一种与已充分表征的高Km酶不同的低Km碘甲状腺原氨酸5'-脱碘酶一致，并表明在游离T4的循环水平下，假定的低Km酶可能具有生理重要性。
