Department of Chemistry, University of Western Ontario, London, ON, Canada N6A 5B7.
J Inorg Biochem. 2010 Mar;104(3):232-44. doi: 10.1016/j.jinorgbio.2009.12.007. Epub 2009 Dec 13.
Metallothionein (MT) is a prominent metal-binding protein and in mammalian systems contains a two-domain betaalpha motif, while in lower life forms MT often consists of only a single-domain structure. There are also unusual MTs from American oysters that consist of multiple domains (from one to four alpha domains). This report details the study of the As(3+)-metalation to two different concatenated triple beta and alpha domain MTs using time-resolved electrospray ionization mass spectrometry (ESI MS). Analysis of kinetic ESI MS data show that alphaalphaalpha human MT and betabetabeta human MT bind As(3+) in a noncooperative manner and involves up to 11 sequential bimolecular reactions. We report the complete progress of the reactions for the As(3+)-metalation of both triple-domain MTs from zero and up to 9 (betabetabeta) or 10As(3+) ions (alphaalphaalpha). The rate constants for the As(3+)-metalation are reported for both the betabetabeta and alphaalphaalpha human MT. At room temperature (298K) and pH3.5, the sequential individual rate constants, k(n) (n=1-9) for the As(3+)-metalation of betabetabetahMT starting at k(1betabetabeta) are 40, 36, 37, 26, 27, 17, 12, 6, and 1M(-1)s(-1); while at room temperature (298K) and pH3.5, the sequential individual rate constants, k(n) (n=1-10) for the As(3+)-metalation of alphaalphaalphahMT starting at k(1alphaalphaalpha) are 52, 45, 46, 42, 38, 36, 29, 25, 14, and 6M(-1)s(-1). The trend in the rate constant values reported for these two triple-domain MT proteins supports our previous proposal that the rate constant values are proportionally related to the total number of equivalent binding sites. The rate of binding for the 1st As(3+) is the fastest we have determined for any MT to date. Additionally, we propose that the data show for the first time for any MT species, that interdomain metalation occurs in the binding of the 10th and 11th As(3+) to alphaalphaalphahMT.
金属硫蛋白（MT）是一种重要的金属结合蛋白，在哺乳动物系统中包含一个双域βα结构，而在较低的生命形式中，MT 通常由单个结构域组成。美国牡蛎中也存在一些不寻常的 MT，它们由多个结构域（一个到四个α结构域）组成。本报告详细介绍了使用时间分辨电喷雾电离质谱（ESI-MS）研究两种不同串联的三β和α结构域 MT 与 As（3+）的金属化。动力学 ESI-MS 数据分析表明，ααα人 MT 和 βββ人 MT 以非协同方式结合 As（3+），涉及多达 11 个顺序双分子反应。我们报告了两种三结构域 MT 从零到 9（βββ）或 10As（3+）离子（ααα）的 As（3+）金属化反应的完整进展。报告了βββ和ααα人 MT 的 As（3+）金属化的速率常数。在室温（298K）和 pH3.5 下，βββ MT 起始于 k（1βββ）的 As（3+）金属化的顺序单个速率常数 k（n）（n=1-9）为 40、36、37、26、27、17、12、6 和 1M（-1）s（-1）；而在室温（298K）和 pH3.5 下，ααα MT 起始于 k（1ααα）的 As（3+）金属化的顺序单个速率常数 k（n）（n=1-10）为 52、45、46、42、38、36、29、25、14 和 6M（-1）s（-1）。这两种三结构域 MT 蛋白报道的速率常数值的趋势支持了我们之前的假设，即速率常数值与等效结合位点的总数成比例。第 1 个 As（3+）的结合速度是我们迄今为止确定的任何 MT 中最快的。此外，我们提出数据首次表明，对于任何 MT 物种，在第 10 和第 11 个 As（3+）与ααα MT 的结合中发生了结构域间金属化。
