On the Mechanism of a Polyunsaturated Fatty Acid Double Bond Isomerase from Propionibacterium acnes
On the Mechanism of a Polyunsaturated Fatty Acid Double Bond Isomerase from Propionibacterium acnes
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DOI：
10.1074/jbc.m809060200
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发表时间：
2009-03-20
影响因子：
4.8
通讯作者：
Feussner, Ivo
中科院分区：
文献类型：
--
作者：
Liavonchanka, Alena;Rudolph, Markus G.;Feussner, Ivo
通过动力学、光谱和热力学研究探讨了痤疮丙酸杆菌多不饱和脂肪酸异构酶(PAI)的催化机理。PAI催化的双键异构化是通过选择性地从C-11上脱除PRO-R氢，然后将这些氢表面转移到C-9上进行的，如C-9-氚底物同位素的转化所示。中点电位、光还原和辅因子置换的数据表明，PAI是通过离子机制作用的，形成FADH(2)和亚油酸碳正离子作为中间产物。根据这一建议，既没有通过停止流动吸收检测到自由基中间体，也没有通过EPR光谱检测到。底物对游离脂肪酸的选择性是由Arg-88和Phe-193之间的相互作用决定的，而这些氨基酸的取代对反应速度有很大的影响，这表明氢转移的效率依赖于游离羧基和FAD的N-5原子之间的固定距离。结合从结构研究和实验中获得的PAI数据，表明多不饱和脂肪酸双键异构酶中至少存在两种不同的原型活性部位几何构型。
The catalytic mechanism of Propionibacterium acnes polyunsaturated fatty acid isomerase (PAI) is explored by kinetic, spectroscopic, and thermodynamic studies. The PAI-catalyzed double bond isomerization takes place by selective removal of the pro-R hydrogen from C-11 followed by suprafacial transfer of this hydrogen to C-9 as shown by conversion of C-9-deuterated substrate isotopologs. Data on the midpoint potential, photoreduction, and cofactor replacement suggest PAI to operate via an ionic mechanism with the formation of FADH(2) and linoleic acid carbocation as intermediates. In line with this proposal, no radical intermediates were detected neither by stopped flow absorption nor by EPR spectroscopy. The substrate preference toward free fatty acids is determined by the interaction between Arg-88 and Phe-193, and the reaction rate is strongly affected by replacement of these amino acids, indicating that the efficiency of the hydrogen transfer relies on a fixed distance between the free carboxyl group and the N-5 atom of FAD. Combining data obtained for PAI from the structural studies and experiments described here suggests that at least two different prototypical active site geometries exist among polyunsaturated fatty acid double bond isomerases.