On the relationships of substrate orientation, hydrogen abstraction, and product stereochemistry in single and double dioxygenations by soybean lipoxygenase-1 and its Ala542Gly mutant
On the relationships of substrate orientation, hydrogen abstraction, and product stereochemistry in single and double dioxygenations by soybean lipoxygenase-1 and its Ala542Gly mutant
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DOI：
10.1074/jbc.m504870200
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发表时间：
2005-11-18
影响因子：
4.8
通讯作者：
Brash, AR
中科院分区：
文献类型：
--
作者：
Coffa, G;Imber, AN;Brash, AR
最近的研究发现，在脂氧合酶（LOX）催化中，立体化学的控制与一个保守的活性位点丙氨酸（S构型过氧化氢产物）或一个相应的甘氨酸（R构型）有关。为了进一步阐明这种立体控制的机制基础，我们比较了大豆LOX-1和将亚油酸转化为13S和9R构型过氧化氢产物的Ala542Gly突变体的起始氢提取的立体选择性。使用11R- h -3和11s - h -3标记的亚油酸底物来检测初始氢提取，我们发现所有的初级过氧化氢产物都是由底物C-11的高度立体选择性的前s氢提取（97 - 99%的前s选择性）形成的。这有力地表明，9R和13S氧合发生在活性位点底物的相同结合取向下，并且由于等效的9R和13S产物是由一个大体积的酯衍生物（1-棕榈酰-2-亚油基磷脂酰胆碱）形成的，因此可以推断取向是尾朝前的。R产物诱导的Ala-Gly突变改变了EPR谱和反应动力学，表明这种Ala-Gly取代的实质性影响延伸到活性位点铁的环境。为了研究底物结合的反向方向，我们研究了Ala542Gly突变体大豆LOX-1对花生四烯酸15s过氧化氢的氧化作用。除了常见的5S、15S-和8S、15S-二氢过氧化物外，形成了新的产物，并通过高效液相色谱、紫外、气相色谱-质谱和核磁共振鉴定为9R、15S-二氢过氧化物二十代5z、7E、11Z、13e -四烯酸，即正常5S、15S产物的R构型“伙伴”。这提供了证据，表明底物的尾部优先结合和羧酸末端优先结合在相同活性位点的产物形成中都与S或R伙伴关系有关。
Recent findings associate the control of stereochemistry in lipoxygenase (LOX) catalysis with a conserved active site alanine for S configuration hydroperoxide products, or a corresponding glycine for R stereoconfiguration. To further elucidate the mechanistic basis for this stereocontrol we compared the stereoselectivity of the initiating hydrogen abstraction in soybean LOX-1 and an Ala542Gly mutant that converts linoleic acid to both 13S and 9R configuration hydroperoxide products. Using 11R- H-3-and 11S-H-3-labeled linoleic acid substrates to examine the initial hydrogen abstraction, we found that all the primary hydroperoxide products were formed with an identical and highly stereoselective pro-S hydrogen abstraction from C-11 of the substrate ( 97 - 99% pro-S-selective). This strongly suggests that 9R and 13S oxygenations occur with the same binding orientation of substrate in the active site, and as the equivalent 9R and 13S products were formed from a bulky ester derivative (1-palmitoyl-2-linoleoylphosphatidylcholine), one can infer that the orientation is tail-first. Both the EPR spectrum and the reaction kinetics were altered by the R product-inducing Ala-Gly mutation, indicating a substantial influence of this Ala-Gly substitution extending to the environment of the active site iron. To examine also the reversed orientation of substrate binding, we studied oxygenation of the 15S-hydroperoxide of arachidonic acid by the Ala542Gly mutant soybean LOX-1. In addition to the usual 5S, 15S- and 8S, 15S-dihydroperoxides, a new product was formed and identified by high-performance liquid chromatography, UV, gas chromatography-mass spectrometry, and NMR as 9R, 15S-dihydroperoxyeicosa5Z, 7E, 11Z, 13E-tetraenoic acid, the R configuration "partner" of the normal 5S, 15S product. This provides evidence that both tail-first and carboxylate end-first binding of substrate can be associated with S or R partnerships in product formation in the same active site.