Doerge D R, Taurog A, Dorris M L
National Center for Toxicological Research, Jefferson, Arkansas 72079-9502.
Arch Biochem Biophys. 1994 Nov 15;315(1):90-9. doi: 10.1006/abbi.1994.1475.
Single turnover experiments were performed with horseradish peroxidase (HRP) to study the mechanism of peroxidase-catalyzed coupling and its stimulation by low concentrations of free diiodotyrosine (DIT). HRP was used because, unlike thyroid peroxidase (TPO) and lactoperoxidase (LPO), the spectral properties of compounds I and II are readily distinguishable. This made it possible to correlate the kinetics and stoichiometry of T4 + T3 formation with spectral data. Incubation of 2 microM preformed HRP-I with 2 microM [125I]Tg (thyroglobulin of low hormone content, high iodotyrosine content) in the presence of 1 microM free DIT yielded about 0.8 residue T4 and 0.2 residue T3 per molecule of Tg. This represents the theoretical maximum for iodothyronine formation, indicating remarkably efficient use of the oxidizing equivalents in HRP-I for coupling. The time course for formation of T4 + T3 was biphasic. During a rapid initial phase (about 1 min), HRP-I was completely converted to HRP-II, coincident with the formation of about 0.65 residues of T4 + T3. During the second slower phase, lasting 10-15 min, HRP-II was completely reduced to the native enzyme, with formation of the remaining T4 + T3. In the absence of DIT, the coupling yield was reduced to 0.5-0.6 residue T4 + T3 per molecule Tg, and the reaction, although considerably slower, was still biphasic. The rapid phase again corresponded to the conversion of HRP-I to HRP-II, and the slower phase to the conversion of HRP-II to native enzyme. To gain insight into the mechanism of the stimulatory effect of free DIT on coupling, we studied the reaction of DIT with HRP-I and HRP-II. Free DIT reacted with both HRP-I and HRP-II in one-electron transfer reactions, and the time course for these reductions resembled those observed with DIT + Tg. These observations suggest that in DIT-stimulated coupling, free DIT radicals act as a shuttle for transferring oxidizing equivalents from the peroxidase intermediates to the DIT residues in Tg. The remarkable efficiency of the HRP-I-mediated coupling reaction implies that (i) only hormonogenic residues in Tg are oxidized and (ii) oxidation of two hormonogenic residues occurs within the same molecule of Tg. A scheme which attempts to explain both kinetic and stoichiometric features of the coupling reaction observed in this study is proposed. This scheme is based on a radical mechanism, consistent with the conclusions reached in the companion paper.
利用辣根过氧化物酶（HRP）进行单周转实验，以研究过氧化物酶催化偶联的机制及其受低浓度游离二碘酪氨酸（DIT）刺激的情况。使用HRP是因为与甲状腺过氧化物酶（TPO）和乳过氧化物酶（LPO）不同，化合物I和II的光谱特性易于区分。这使得将T4 + T3形成的动力学和化学计量与光谱数据相关联成为可能。在1 microM游离DIT存在的情况下，将2 microM预先形成的HRP-I与2 microM [125I]Tg（低激素含量、高碘酪氨酸含量的甲状腺球蛋白）一起孵育，每分子Tg产生约0.8个T4残基和0.2个T3残基。这代表了碘甲状腺原氨酸形成的理论最大值，表明HRP-I中的氧化当量在偶联中得到了显著有效的利用。T4 + T3形成的时间进程是双相的。在快速的初始阶段（约1分钟），HRP-I完全转化为HRP-II，同时形成约0.65个T4 + T3残基。在持续10 - 15分钟的第二个较慢阶段，HRP-II完全还原为天然酶，同时形成其余的T4 + T3。在没有DIT的情况下，偶联产率降至每分子Tg 0.5 - 0.6个T4 + T3残基，并且反应虽然相当慢，但仍然是双相的。快速阶段再次对应于HRP-I向HRP-II的转化，较慢阶段对应于HRP-II向天然酶的转化。为了深入了解游离DIT对偶联的刺激作用机制，我们研究了DIT与HRP-I和HRP-II的反应。游离DIT在单电子转移反应中与HRP-I和HRP-II都发生反应，这些还原反应的时间进程与DIT + Tg观察到的相似。这些观察结果表明，在DIT刺激的偶联中，游离DIT自由基充当了将氧化当量从过氧化物酶中间体转移到Tg中DIT残基的穿梭体。HRP-I介导的偶联反应的显著效率意味着：（i）Tg中只有激素生成残基被氧化；（ii）两个激素生成残基的氧化发生在同一Tg分子内。本文提出了一个试图解释本研究中观察到的偶联反应的动力学和化学计量特征的方案。该方案基于自由基机制，与 companion paper中得出的结论一致。
