Alternative routes for entry of HgX2 into the active site of mercuric ion reductase depend on the nature of the X ligands
Alternative routes for entry of HgX2 into the active site of mercuric ion reductase depend on the nature of the X ligands
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DOI：
10.1021/bi982680c
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发表时间：
1999-03-23
期刊：
影响因子：
2.9
通讯作者：
Miller, SM
中科院分区：
文献类型：
--
作者：
Engst, S;Miller, SM
野生型汞离子还原酶（CCCC 酶）的每个 Hg(II) 结合位点均具有四个半胱氨酸、一个氧化还原活性对和一个 C 末端对。 C 末端半胱氨酸突变为丙氨酸（CCAA 酶）会导致使用 Hg(SR)(2) 底物的稳态汞离子还原酶活性丧失。然而，CCCC 和 CCAA 酶在前稳态条件下使用 HgBr2 作为底物表现出同样高的结合率和周转率 [Engst 和 Miller (1998) Biochemistry 37, 11496-11507.]。由于这些 HgX2 底物中的配体在大小和对 Hg(II) 的亲和力方面都不同，因此这些特性中的一种或两种可能导致它们与 CCAA 酶的不同反应性。为了进一步探讨这两个特性的重要性，我们研究了 CCCC 和 CCAA 与 Hg(CN)(2)（具有小而高亲和力的配体）和 Hg(Cys)(2)（具有大的高亲和力配体）的前稳态反应。结果表明，具有小配体的 HgX2 底物可以在 C 端半胱氨酸不存在的情况下快速接近氧化还原活性半胱氨酸，但具有大配体的 HgX2 底物需要 C 端半胱氨酸才能快速接近。此外，得出的结论是，在 Hg(II) 到达内部活性位点中的氧化还原活性半胱氨酸之前，C 端半胱氨酸在去除高亲和力配体方面发挥着关键作用，因为 HgX2 底物与高亲和力配体的直接接触会导致形成受抑制的复合物。与结果一致，在来自芽孢杆菌属的酶的结构中可以鉴定出直接通向氧化还原活性半胱氨酸的窄通道和通过与C端半胱氨酸的初始接触通向氧化还原活性半胱氨酸的较宽通道。 RC607。
Wild-type mercuric ion reductase (CCCC enzyme) possesses four cysteines in each of its Hg(II) binding sites, a redox-active pair and a C-terminal pair. Mutation of the C-terminal cysteines to alanines (CCAA enzyme) leads to a loss of steady-state mercuric ion reductase activity using Hg(SR)(2) substrates. However, CCCC and CCAA enzymes exhibit an equally high rate of binding and turnover using HgBr2 as substrate under pre-steady-state conditions [Engst and Miller (1998) Biochemistry 37, 11496-11507.]. Since the ligands in these HgX2 substrates differ both in size and in affinity for Hg(II), one or both of these properties may contribute to their different reactivities with CCAA enzyme. To further explore the importance of these two properties, we have examined the pre-steady-state reactions of CCCC and CCAA with Hg(CN)(2), which has small, high-affinity ligands, and with Hg(Cys)(2), which has bulky, high-affinity ligands. The results indicate that HgX2 substrates with small ligands can rapidly access the redox-active cysteines in the absence of the C-terminal cysteines, but those with large ligands require the C-terminal cysteines for rapid access. In addition, it is concluded that the C-terminal cysteines play a critical role in removing the high-affinity ligands before Hg(II) reaches the redox-active cysteines in the inner active site, since direct access of HgX2 substrates with high-affinity ligands leads to formation of an inhibited complex. Consistent with the results, both a narrow channel leading directly to the redox-active cysteines and a wider channel leading to the redox-active cysteines via initial contact with the C-terminal cysteines can be identified in the structure of the enzyme from Bacillus sp. RC607.