Fitzgerald M C, Chernushevich I, Standing K G, Whitman C P, Kent S B
The Scripps Research Institute, La Jolla, CA 92037, USA.
Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):6851-6. doi: 10.1073/pnas.93.14.6851.
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: (desPro1)4OT, a truncated construct in which Pro1 was deleted; (Cpc1)4OT in which Pro1 was replaced with cyclopentane carboxylate; a derivative [Met(O)45]4OT in which Met45 was oxidized to the sulfoxide; and an analogue (Nle45)4OT in which Met45 was replaced with norleucine. ESI of (Nle45)4OT, (Cpc1)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 (desPro1)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 Pro1 residue in 4OT.
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）及其通过全化学合成制备的四种类似物的四级结构。野生型4OT是由62个氨基酸亚基组成的六聚体，不含半胱氨酸残基。这些类似物分别为：（desPro1）4OT，一种缺失Pro1的截短构建体；（Cpc1）4OT，其中Pro1被环戊烷羧酸取代；衍生物[Met（O）45]4OT，其中Met45被氧化为亚砜；以及类似物（Nle45）4OT，其中Met45被正亮氨酸取代。在天然酶完全活性的溶液条件下（5 mM碳酸氢铵缓冲液，pH 7.5），对（Nle45）4OT、（Cpc1）4OT和4OT进行电喷雾电离，飞行时间质谱检测到的主要物种为完整的六聚体。相比之下，在类似条件下对[Met（O）45]4OT和（desPro1）4OT进行分析，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。结合为4OT及其这些类似物收集的动力学数据，ESI-TOF质谱法还为4OT中具有催化重要性的Pro1残基的结构和机制意义提供了重要证据。
