Zinc content of Escherichia coli-expressed constitutive isoforms of nitric-oxide synthase -: Enzymatic activity and effect of pterin
Zinc content of Escherichia coli-expressed constitutive isoforms of nitric-oxide synthase -: Enzymatic activity and effect of pterin
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DOI：
10.1074/jbc.274.21.14537
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发表时间：
1999-05-21
影响因子：
4.8
通讯作者：
Masters, BSS
中科院分区：
文献类型：
--
作者：
Miller, RT;Martásek, P;Masters, BSS
最近，我们获得了 X 射线晶体学数据，显示大肠杆菌表达的牛内皮一氧化氮合酶 (eNOS) 和大鼠神经元一氧化氮合酶 (nNOS) 的结构中存在 ZnS4 中心，锌原子由两个 CXXXXC 基序协调，每个 NOS 单体贡献一个基序（大鼠 nNOS 中的半胱氨酸 326 到半胱氨酸 331），突变nNOS 半胱氨酸 331 转变为丙氨酸 (C331A) 导致 NO 丢失。合成活性，还导致无法有效结合锌。虽然 nNOS C331A 突变体与高浓度 L-精氨酸长时间孵育会产生催化活性酶，但锌结合不会恢复。在这项研究中，我们使用螯合测定和电热蒸发-电感耦合等离子体质谱法研究了野生型 nNOS 和 eNOS 以及 C331A 突变的 nNOS 中的锌化学计量。数据显示，在充满 (6R)-5,6,7,8-四氢-L-生物蝶呤的野生型 nNOS 和 eNOS 中，血红素与锌的化学计量约为 2:1，并表明重新激活的 nNOS C331A 突变体结合锌的能力有限。本研究证实，NOS 中的锌是结构性的，而不是催化性的，对于维持最佳功能、酶活性、组成型 NOS 很重要。
Recently, we obtained x-ray crystallographic data showing the presence of a ZnS4 center in the structure of Escherichia coli-expressed bovine endothelial nitric-oxide synthase (eNOS) and rat neuronal nitric oxide synthase (nNOS), The zinc atom is coordinated by two CXXXXC motifs, one motif being contributed by each NOS monomer (cysteine 326 through cysteine 331 in rat nNOS), Mutation of the nNOS cysteine 331 to alanine (C331A) results in the loss of NO. synthetic activity and also results in an inability to bind zinc efficiently. Although prolonged incubation of the C331A mutant of nNOS with high concentrations of L-arginine results in a catalytically active enzyme, zinc binding is not restored. In this study, we investigate the zinc stoichiometry in wild-type nNOS and eNOS, as well as in the C331A-mutated nNOS, using a chelation assay and electrothermal vaporization-inductively coupled plasma-mass spectrometry. The data reveal an approximate 2:1 stoichiometry of heme to zinc in (6R)-5,6,7,8-tetrahydro-L-biopterin-replete, wild-type nNOS and eNOS and show that the reactivated C331A mutant of nNOS has a limited ability to bind zinc. The present study substantiates that the zinc in NOS is structural rather than catalytic and is important for maintaining optimally functional, enzymatically active, constitutive NOSs.