Membrane association of and critical residues in the catalytic domain of human neuropathy target esterase
Membrane association of and critical residues in the catalytic domain of human neuropathy target esterase
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DOI：
10.1074/jbc.m002921200
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发表时间：
2000-08-11
影响因子：
4.8
通讯作者：
Glynn, P
中科院分区：
文献类型：
--
作者：
Atkins, J;Glynn, P
神经病变靶酯酶（NTE）是脊椎动物神经元中一种完整的膜蛋白，也是一种新的丝氨酸水解酶家族的成员。在这里，我们发现在大肠杆菌中表达的重组多肽NEST与酯底物和共价抑制剂的反应方式与NTE非常相似，NEST包含人类NTE的727-1216残基，位点定向诱变显示丝氨酸966和两个天冬氨酸残基Asp（1086）和Asp（960）对催化作用至关重要。通过与[H-3]氟磷酸二异丙基反应，确定了活性位点丝氨酸为Ser(966)，并获得了Ser（966）加合物向天冬氨酸残基转移异丙基的证据。在大肠杆菌裂解物和纯化过程中，都需要洗涤剂来溶解NEST。洗涤剂提取物的催化活性丧失，但当纯化的NEST被纳入二油基磷脂酰胆碱脂体时，催化活性恢复。水亲和分析未表明在NEST序列中存在跨膜片段。然而，包括洗涤相分离实验和脂质体结合的NEST对蛋白质水解的抗性在内的生化证据表明，与大多数真核丝氨酸水解酶不同，NTE的催化结构域具有完整的膜蛋白性质。
Neuropathy target esterase (NTE) is an integral membrane protein in vertebrate neurons and a member of a novel family of putative serine hydrolases. Here we show that NEST, a recombinant polypeptide expressed in Escherichia coli, reacts with an ester substrate and covalent inhibitors in a manner very similar to NTE, NEST comprises residues 727-1216 of human NTE, and site-directed mutagenesis revealed that serine 966 and two aspartate residues, Asp(1086) and Asp(960), are critical for catalysis. The results of mutating the 11 histidines in NEST suggest that NTE does not use a conventional catalytic triad, By reacting NEST with [H-3]diisopropyl fluorophosphate, Ser(966) was confirmed as the active-site serine, and evidence was obtained that an isopropyl group is transferred from the Ser(966) adduct to an aspartate residue. Detergent was required both for solubilization of NEST from lysates of E, coli and during purification procedures. Catalytic activity was lost in detergent extracts, but was restored when purified NEST was incorporated into dioleoylphosphatidylcholine liposomes, Hydropathy analysis did not indicate the presence of membrane-spanning segments within the NEST sequence. However, biochemical evidence including detergent-phase separation experiments and the resistance of liposome-incorporated NEST to proteolysis indicated that, unlike most eukaryotic serine hydrolases, the catalytic domain of NTE has integral membrane protein properties.