Smith Sarah J, Casellato Annelise, Hadler Kieran S, Mitić Natasa, Riley Mark J, Bortoluzzi Adailton J, Szpoganicz Bruno, Schenk Gerhard, Neves Ademir, Gahan Lawrence R
School of Molecular and Microbial Sciences, The University of Queensland, St Lucia, QLD, Australia.
J Biol Inorg Chem. 2007 Nov;12(8):1207-20. doi: 10.1007/s00775-007-0286-y. Epub 2007 Aug 15.
Purple acid phosphatase from pig uterine fluid (uteroferrin), a representative of the diverse family of binuclear metallohydrolases, requires a heterovalent Fe(III)Fe(II) center for catalytic activity. The active-site structure and reaction mechanism of this enzyme were probed with a combination of methods including metal ion replacement and biomimetic studies. Specifically, the asymmetric ligand 2-bis{[(2-pyridylmethyl)-aminomethyl]-6-[(2-hydroxybenzyl)(2-pyridylmethyl)]aminomethyl}-4-methylphenol and two symmetric analogues that contain the softer and harder sites of the asymmetric unit were employed to assess the site selectivity of the trivalent and divalent metal ions using (71)Ga NMR, mass spectrometry and X-ray crystallography. An exclusive preference of the harder site of the asymmetric ligand for the trivalent metal ion was observed. Comparison of the reactivities of the biomimetics with Ga(III)Zn(II) and Fe(III)Zn(II) centers indicates a higher turnover for the former, suggesting that the M(III)-bound hydroxide acts as the reaction-initiating nucleophile. Catalytically active Ga(III)Zn(II) and Fe(III)Zn(II) derivatives were also generated in the active site of uteroferrin. As in the case of the biomimetics, the Ga(III) derivative has increased reactivity, and a comparison of the pH dependence of the catalytic parameters of native uteroferrin and its metal ion derivatives supports a flexible mechanistic strategy whereby both the mu-(hydr)oxide and the terminal M(III)-bound hydroxide can act as nucleophiles, depending on the metal ion composition, the geometry of the second coordination sphere and the substrate.
猪子宫液中的紫色酸性磷酸酶（子宫铁蛋白）是双核金属水解酶多样家族的代表，其催化活性需要一个异价的Fe(III)Fe(II)中心。通过包括金属离子置换和仿生研究在内的多种方法相结合，对该酶的活性位点结构和反应机制进行了探究。具体而言，使用不对称配体2-双{[(2-吡啶甲基)-氨基甲基]-6-[(2-羟基苄基)(2-吡啶甲基)]氨基甲基}-4-甲基苯酚以及两个包含不对称单元较软和较硬位点的对称类似物，利用(71)Ga NMR、质谱和X射线晶体学来评估三价和二价金属离子的位点选择性。观察到不对称配体较硬的位点对三价金属离子具有排他性偏好。对具有Ga(III)Zn(II)和Fe(III)Zn(II)中心的仿生化合物反应活性的比较表明，前者的周转数更高，这表明与M(III)结合的氢氧化物充当反应起始亲核试剂。在子宫铁蛋白的活性位点也生成了具有催化活性的Ga(III)Zn(II)和Fe(III)Zn(II)衍生物。与仿生化合物的情况一样，Ga(III)衍生物的反应活性有所增加，对天然子宫铁蛋白及其金属离子衍生物催化参数的pH依赖性比较支持了一种灵活的机制策略，即根据金属离子组成、第二配位层的几何结构和底物的不同，μ-(氢)氧化物和末端与M(III)结合的氢氧化物都可以充当亲核试剂。
