Probing the oligomeric structure of an enzyme by electrospray ionization time-of-flight mass spectrometry
Probing the oligomeric structure of an enzyme by electrospray ionization time-of-flight mass spectrometry
复制标题
DOI：
10.1073/pnas.93.14.6851
复制
发表时间：
1996-07-09
影响因子：
11.1
通讯作者：
Kent, SBH
中科院分区：
文献类型：
--
作者：
Fitzgerald, MC;Chernushevich, I;Kent, SBH
采用电喷雾电离飞行时间（ESI-TOF）质谱法研究了4-草酰巴豆酸互变异构酶（EC 5.3.2；4OT）的四级结构，并通过全化学合成制备了四种类似物，野生型4OT是62个氨基酸亚基的六聚体，不含半胱氨酸残基。类似物是：(desPro(1))4OT，一种截短的构建体，其中 Pro(1) 被删除； (Cpc(1))40T，其中Pro(1)被环戊烷羧酸酯取代； [Met(O)(45)]4OT衍生物，其中Met(45)被氧化成亚砜；和类似物(Nle(45))4OT，其中Met(45)被正亮氨酸取代。在天然酶完全有活性的溶液条件下（5 mM 碳酸氢铵缓冲液，pH 7.5），对 (Nle(45))4OT、(Cpc(1))4OT 和 4OT 进行 ESI 分析，结果显示完整的六聚体是 TOF 质谱检测到的主要物质。相比之下，在类似条件下分析 [Met(O)(45)]4OT 和 (desPro(1))4OT 主要产生单体离子。 ESI-TOF 测量结果与圆二色光谱获得的结构数据一致。在收集 4OT 和这些类似物的动力学数据的背景下，ESI-TOF 质谱还为 4OT 中催化重要的 Pro(1) 残基的结构和机械意义提供了重要证据。
Electrospray ionization time-of-flight (ESI-TOF) mass spectrometry was used to study the quaternary structure of 4-oxalocrotonate tautomerase (EC 5.3.2; 4OT), and four analogues prepared by total chemical synthesis, Wild-type 4OT is a hexamer of 62 amino acid subunits and contains no cysteine residues. The analogues were: (desPro(1))4OT, a truncated construct in which Pro(1) was deleted; (Cpc(1))40T in which Pro(1) was replaced with cyclopentane carboxylate; a derivative [Met(O)(45)]4OT in which Met(45) was oxidized to the sulfoxide; and an analogue (Nle(45))4OT in which Met(45) was replaced with norleucine. ESI of (Nle(45))4OT, (Cpc(1))4OT, and 4OT from solution conditions under which the native enzyme was fully active (5 mM ammonium bicarbonate buffer, pH 7.5) gave the intact hexamer as the major species detected by TOF mass spectrometry, In contrast, analysis of [Met(O)(45)]4OT and (desPro(1))4OT under similar conditions yielded predominantly monomer ions. The ESI-TOF measurements were consistent with structural data obtained from circular dichroism spectroscopy. In the context of kinetic data collected for 4OT and these analogues, ESI-TOF mass spectrometry also provided important evidence for the structural and mechanistic significance of the catalytically important Pro(1) residue in 4OT.