Substrate positioning by His92 is important in catalysis by purple acid phosphatase
Substrate positioning by His92 is important in catalysis by purple acid phosphatase
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DOI：
10.1111/j.1742-4658.2005.04686.x
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发表时间：
2005-06-01
期刊：
影响因子：
5.4
通讯作者：
Averill, BA
中科院分区：
文献类型：
--
作者：
Funhoff, EG;Wang, WL;Averill, BA
单多肽哺乳动物紫色酸性磷酸酶 (PAP) 的蛋白水解导致环残基 Asp146 与活性位点残基 Asn91 和/或 His92 之间相互作用的丧失。虽然 Asn91 是混合价二铁中心的二价金属的配体，但 His92 在催化机制中的作用尚不清楚。对 His92 进行定点诱变以检查该残基在单多肽 PAP 中的作用。 His92 转化为 Ala，消除了该残基与活性位点的极性相互作用，导致在最佳 pH 下催化活性降低 10 倍。相反，将该残基转化为 Asn（既不能充当质子供体也不能充当受体，但可以提供氢键相互作用）导致在最佳 pH 下活性增加三倍。两种突变酶都具有更酸性的最适 pH，pK(es,1) 值与催化过程中氢氧化铁 (III) 单元或第二配位层中的氢氧化物的参与一致。这些结果与 EPR 数据一起支持 His92 在定位亲核试剂或底物中的作用，而不是直接在酸或碱催化中发挥作用。广泛的氢键网络的存在可以微调 His92 的位置，这与这一提议是一致的。
Proteolysis of single polypeptide mammalian purple acid phosphatases (PAPs) results in the loss of an interaction between the loop residue Asp146 and the active site residues Asn91 and/or His92. While Asn91 is a ligand to the divalent metal of the mixed-valent di-iron center, the role of His92 in the catalytic mechanism is unknown. Site-directed mutagenesis of His92 was performed to examine the role of this residue in single polypeptide PAP. Conversion of His92 into Ala, which eliminates polar interactions of this residue with the active site, resulted in a 10-fold decrease in catalytic activity at the optimal pH. Conversely, conversion of this residue into Asn, which cannot function as either a proton donor or acceptor, but can provide hydrogen-bonding interactions, resulted in a three-fold increase in activity at the optimal pH. Both mutant enzymes had more acidic pH optima, with pK(es,1) values consistent with the involvement of an iron(III) hydroxide unit or a hydroxide in the second coordination sphere in catalysis. These results, together with EPR data, support a role of His92 in positioning either the nucleophile or the substrate, rather than directly in acid or base catalysis. The existence of an extensive hydrogen-bonding network that could fine-tune the position of His92 is consistent with this proposal.