Arsenic binding to human metallothionein
Arsenic binding to human metallothionein
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DOI：
10.1021/ja062914c
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发表时间：
2006-09-27
影响因子：
15
通讯作者：
Stillman, Martin J.
中科院分区：
文献类型：
--
作者：
Ngu, Thanh T.;Stillman, Martin J.
全世界报告的慢性砷中毒病例数量正在上升，因此研究砷的长期影响至关重要。As3+很容易与生物硫醇结合，包括哺乳动物金属硫蛋白（MT），这是一种普遍存在的富含硫的金属蛋白，已知可以协调多种金属。该双结构域哺乳动物蛋白将二价金属(M)结合成两个具有M3Scys9 （β）和M4Scys11 （α）化学计量的金属硫酸酯簇。我们报道了As3+与As3Scys9 （β）和As3Scys11 （α）的化学计量结合到重组人金属硫蛋白（rhMT）亚型1a蛋白上。此外，我们报道了rhMT与As3+的α和β域饱和反应的完整动力学分析。金属化反应中的物种形成是用时间和温度分辨的电喷雾质谱法测定的。As3+与α和β MT结构域的结合反应是非合作的，涉及三个连续的双分子金属化步骤。该分析首次完整模拟了MT逐步金属化反应的实验数据，显示了无金属MT、载子MT和每种as -MT中间物质的相对浓度随时间和温度的变化。在室温（298 K）和pH为3.5的条件下，As3+与apo-RMT结合的速率常数分别为5.5、6.3和3.9 M-1 s(-1)，而与apo- β - MT结合的速率常数分别为3.6、2.0和0.6 M-1 s（-1）。as -1- h -6- β MT的生成活化能为32 kJ mol(-1)， as -2- h -3- β MT为35 kJ mol(-1)， as -3- β MT为29 kJ mol(-1)， as -1- h -8- α MT为33 kJ mol(-1)， as -2- h -5- α MT为29 kJ mol(-1)， as -3- h -2- α MT为23 kJ mol（-1）。
The number of reported cases of chronic arsenic poisoning is on the rise throughout the world, making the study of the long-term effects of arsenic critical. As3+ binds readily to biological thiols, including mammalian metallothionein (MT), which is an ubiquitous sulfur-rich metalloprotein known to coordinate a wide range of metals. The two-domain mammalian protein binds divalent metals (M) into two metal-thiolate clusters with stoichiometries of M3Scys9 (beta) and M4Scys11 (alpha). We report that As3+ binds with stoichiometries of As3Scys9 (beta) and As3Scys11 (alpha) to the recombinant human metallothionein (rhMT) isoform 1a protein. Further, we report the complete kinetic analysis of the saturation reactions of the separate alpha and beta domains of rhMT with As3+. Speciation in the metalation reactions was determined using time- and temperature-resolved electrospray ionization mass spectrometry. The binding reaction of As3+ to the alpha and beta MT domains is shown to be noncooperative and involves three sequential, bimolecular metalation steps. The analyses allow for the first time the complete simulation of the experimental data for the stepwise metalation reaction of MT showing the relative concentrations of the metal-free, apo MT and each of the As-MT intermediate species as a function of time and temperature. At room temperature (298 K) and pH 3.5, the individual rate constants for the first, second, and third As3+ binding to apo-RMT are 5.5, 6.3, and 3.9 M-1 s(-1) and for apo-beta MT the constants are 3.6, 2.0, and 0.6 M-1 s(-1). The activation energy for formation of As-1-H-6-beta MT is 32 kJ mol(-1), for As-2-H-3-beta MT it is 35 kJ mol(-1), for As-3-beta MT it is 29 kJ mol(-1), for As-1-H-8-alpha MT it is 33 kJ mol(-1), for As-2-H-5-alpha MT it is 29 kJ mol(-1), and for As-3-H-2-alpha MT it is 23 kJ mol(-1).