Pande Abhay H, Moe David, Nemec Kathleen N, Qin Shan, Tan Shuhua, Tatulian Suren A
Biomolecular Science Center, University of Central Florida, Orlando, Florida 32826, USA.
Biochemistry. 2004 Nov 23;43(46):14653-66. doi: 10.1021/bi048775y.
Mammalian 5-lipoxygenase (5-LO) catalyzes the conversion of arachidonic acid (AA) to leukotrienes, potent inflammatory mediators. 5-LO is activated by a Ca(2+)-mediated translocation to membranes, and demonstrates the characteristic features of interfacially activated enzymes, yet the mechanism of membrane binding of 5-LO is not well understood. In an attempt to understand the mechanism of lipid-mediated activation of 5-LO, we have studied the effects of a large set of lipids on human recombinant 5-LO activity, as well as mutual structural effects of 5-LO and membranes. In the presence of 0.35 mM phosphatidylcholine (PC) and 0.2 mM Ca(2+), there was substrate inhibition at >100 microM AA. Data analysis at low AA concentrations yielded the following: K(m) approximately 103 microM and k(cat) approximately 56 s(-1). 5-LO activity was supported by PC more than by any other lipid tested except for a cationic lipid, which was more stimulatory than PC. Binding of 5-LO to zwitterionic and acidic membranes was relatively weak; the extent of binding increased 4-8 times in the presence of Ca(2+), whereas binding to cationic membranes was stronger and essentially Ca(2+)-independent. Polarized attenuated total reflection infrared experiments implied that 5-LO binds to membranes at a defined orientation with the symmetry axis of the putative N-terminal beta-barrel tilted approximately 45 degrees from the membrane normal. Furthermore, membrane binding of 5-LO resulted in dehydration of the membrane surface and was paralleled with stabilization of the structures of both 5-LO and the membrane. Our results provide insight into the understanding of the effects of membrane surface properties on 5-LO-membrane interactions and the interfacial activation of 5-LO.
哺乳动物5-脂氧合酶（5-LO）催化花生四烯酸（AA）转化为白三烯，白三烯是强效的炎症介质。5-LO通过Ca（2+）介导的向膜的转位而被激活，并表现出界面激活酶的特征，但5-LO与膜结合的机制尚不清楚。为了了解脂质介导的5-LO激活机制，我们研究了大量脂质对人重组5-LO活性的影响，以及5-LO与膜的相互结构效应。在存在0.35 mM磷脂酰胆碱（PC）和0.2 mM Ca（2+）的情况下，当AA浓度>100 microM时存在底物抑制。在低AA浓度下的数据分析得出以下结果：K（m）约为103 microM，k（cat）约为56 s（-1）。除阳离子脂质比PC更具刺激性外，PC对5-LO活性的支持作用比其他任何测试脂质都要强。5-LO与两性离子膜和酸性膜的结合相对较弱；在Ca（2+）存在下，结合程度增加4-8倍，而与阳离子膜的结合更强且基本上与Ca（2+）无关。偏振衰减全反射红外实验表明，5-LO以特定方向与膜结合，假定的N端β桶的对称轴与膜法线倾斜约4５度。此外，5-LO与膜的结合导致膜表面脱水，并与5-LO和膜结构的稳定同时发生。我们的结果为理解膜表面性质对5-LO-膜相互作用和5-LO界面激活的影响提供了见解。
