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首页> 外文期刊>The FEBS journal >Substrate positioning by His92 is important in catalysis by purple acid phosphatase
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Substrate positioning by His92 is important in catalysis by purple acid phosphatase

机译：His92对底物的定位在紫色酸性磷酸酶的催化​​中很重要

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摘要

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.
机译：单个多肽哺乳动物紫色酸性磷酸酶（PAP）的蛋白水解作用导致环残基Asp146与活性位点残基Asn91和/或His92之间相互作用的丧失。虽然Asn91是混合二价铁中心二价金属的配体，但His92在催化机理中的作用尚不清楚。进行His92的定点诱变以检查该残基在单个多肽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值下催化活性降低了10倍。相反，该残基向Asn的转化不能充当质子供体或受体，但可以提供氢键相互作用，导致在最佳pH值下活性增加了三倍。两种突变酶均具有更强的酸性pH最佳值，其pK（es，1）值与催化的第二配位球中氢氧化铁（III）单元或氢氧化物的参与一致。这些结果以及EPR数据支持His92在亲核试剂或底物定位中的作用，而不是直接在酸或碱催化中的作用。该提议的存在是广泛的氢键网络的存在，该网络可以微调His92的位置。

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