Use of deuterium kinetic isotope effects to probe the cryptoregiochemistry of Delta 9 desaturation
Use of deuterium kinetic isotope effects to probe the cryptoregiochemistry of Delta 9 desaturation
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DOI：
10.1021/ja953262n
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发表时间：
1996-07-03
影响因子：
15
通讯作者：
Behrouzian, B
中科院分区：
文献类型：
--
作者：
Buist, PH;Behrouzian, B
脂肪酸的生物合成脱氢（去饱和）1，如硬脂酰CoA（1）的Δ9去饱和酶介导的转化为油酰CoA（2）所示，代表了酶选择性的更精湛的表现之一。两类去饱和酶催化这种有趣的转化：可溶性植物酶含有羧酸根桥接的非血红素二铁中心2和不太好表征的非血红素铁膜结合催化剂，如酿酒酵母3和大鼠肝脏中发现的Δ9去饱和酶。4鉴于越来越多的证据表明去饱和酶和羟化酶在蛋白质水平上具有结构相关性，我们采用了这样的观点，即去饱和是由类似于生物羟化的氢提取步骤引发的。方案1中概述了烯烃的一些可能的后续步骤。6.我们将注意力集中在S. cereVisiae，并且已经暂时将推定的铁-氧代氧化物质放置在底物的C-9附近，因为该酶系统始终比相应的10-硫代类似物更有效地氧化9-硫代脂肪酸类似物。12在这篇文章中，我们报告了一项研究的结果，在这项研究中，我们通过测量每个CH键断裂的氘同位素效应，进一步研究了酵母Δ9去饱和的密码区域化学。13为了加速我们的同位素效应研究，我们决定进行涉及甲基7-硫代硬脂酸-9，9-d2的直接竞争实验。（3-9，9-d2）与7-硫代硬脂酸甲酯（3）和7-硫代硬脂酸甲酯-10，10-d2（3-10，使用硬脂酸甲酯-d2/d 0混合物会使油酸甲酯-d1/d2混合物的分析复杂化。由于内源性（d 0）油酸酯的质谱干扰，生成d 0产物。将硫原子置于位置7以促进氘化底物的合成。我们先前已经表明，7-硫代硬脂酸甲酯（3）转化为相应的硫代油酸酯产物（4）。14
The biological syn-dehydrogenation (desaturation) of fatty acids1 as exemplified by the Δ9 desaturase-mediated transformation of stearoyl CoA (1) to give oleyl CoA (2) represents one of the more virtuosic displays of enzymatic selectivity. Two classes of desaturases catalyze this intriguing transformation: soluble plant enzymes containing a carboxylate-bridged, nonheme diiron center2 and less well-characterized, nonheme iron, membrane-bound catalysts as represented by the Δ9 desaturase found in Saccharomyces cereVisiae3 and rat liver. 4 In light of the mounting evidence5 that desaturases and hydroxylases are structurally related at the protein level, we have adopted the view that desaturations are initiated by a hydrogen abstraction step similar to that proposed for biohydroxylation. Some of the possible subsequent steps to the olefin are outlined in Scheme 1. 6 We have focussed our attention on the Δ9 desaturase of S. cereVisiae and have tentatively placed the putative iron-oxo oxidizing species near C-9 of the substrate since this enzyme system consistently oxygenates 9-thia fatty acid analogues more efficiently than the corresponding 10-thia analogues. 12 In this communication, we report the results of a study in which we further investigate the cryptoregiochemistry of yeast Δ9 desaturation by measuring the deuterium isotope effect for each individual CH bond cleavage. 13 In order to expedite our isotope effect study, we decided to run direct competition experiments involving methyl 7-thiastearate-9, 9-d2 (3-9, 9-d2) vs methyl 7-thiastearate (3) and methyl7-thiastearate-10, 10-d2 (3-10, 10-d2) vs methyl 7-thiastearate (3).Use of methyl stearate-d2/d0 mixtures would have complicated the analysis of the methyl oleate-d1/d0 product due to mass spectral interference by endogenous (d0) oleate. The sulfur atom was placed at position 7 in order to facilitate the synthesis of the deuterated substrates. We have shown previously that methyl 7-thiastearate (3) is converted to the corresponding thiaoleate product (4). 14