McAndrew R S, Chapman K D
Division of Biochemistry and Molecular Biology, Department of Biological Sciences, University of North Texas, Denton, TX 76203-5220, USA.
Biochim Biophys Acta. 1998 Feb 5;1390(1):21-36. doi: 10.1016/s0005-2760(97)00166-5.
An ATP-, Ca2+-, and CoA-independent acyltransferase activity, designated "N-acylphosphatidylethanolamine (NAPE) synthase", was reported to catalyze the direct acylation of phosphatidylethanolamine (PE) with free fatty acids (FFAs) in cottonseed microsomes [K.D. Chapman, T.S. Moore, Jr., Plant Physiol. 102 (3) (1993) 761-769]. Here, NAPE synthase was purified 138, 176-fold from crude cottonseed homogenates to a specific activity of 5.98 mumol min-1 mg-1 protein by immobilized artificial membrane chromatography. Enzyme purity was confirmed by the presence of a 64 kDa polypeptide in fractions analyzed by tricine-SDS-PAGE. Initial velocity measurements with various free fatty acids ([14C]-linoleic, -palmitic, -oleic, -stearic and -myristic acids) and saturating concentrations of dioleoyl-PE revealed non-Michaelis-Menten, biphasic kinetics with high and low affinity sites demonstrating positive cooperativity specific for each [14C]-FFA. In contrast to FFA substrates, no kinetic differences were observed for two different molecular species of PE, (18:1,18:1)-PE and (16:0,18:2)-PE, and biphasic curves were not pronounced. Neither [14C]-dipalmitoylphosphatidylcholine nor [14C]-palmitoyl-CoA served as acyl donors for the synthesis of NAPE, indicating a preference for FFAs as the acyl donor. Also, neither ethanolamine nor sphingosine functioned as acyl acceptor molecule to form N-acylethanolamine or ceramide, respectively, indicating specificity for the phospholipid PE. NAPE synthase was inactivated in a time- and concentration-dependent manner by diisopropylfluorophosphate (DFP) through the apparent modification of one serine residue. Palmitic acid protected the enzyme from DFP-inactivation and [14C]-DFP incorporation, suggesting that a serine residue probably binds FFAs in the enzyme's active site forming an acyl-enzyme intermediate. Collectively, these results provide new information on the kinetic behavior of a purified, integral membrane enzyme which synthesizes a bilayer-stabilizing product from two lipid-soluble substrates. The biochemical properties of cottonseed NAPE synthase are consistent with a possible free fatty acid scavenging role in vivo. (c) 1998 Elsevier Science B.V.
据报道，一种不依赖ATP、Ca2+和辅酶A的酰基转移酶活性，即“N-酰基磷脂酰乙醇胺（NAPE）合酶”，可催化棉籽微粒体中磷脂酰乙醇胺（PE）与游离脂肪酸（FFA）的直接酰化反应[K.D.查普曼，T.S.摩尔，Jr.，《植物生理学》102(3)(1993)761-769]。在此，通过固定化人工膜色谱法从棉籽粗匀浆中纯化出NAPE合酶，纯化倍数达138、176倍，比活性为5.98 μmol min-1 mg-1蛋白质。通过tricine-SDS-PAGE分析的组分中存在一条64 kDa的多肽，证实了酶的纯度。用各种游离脂肪酸（[14C]-亚油酸、-棕榈酸、-油酸、-硬脂酸和-肉豆蔻酸）以及饱和浓度的二油酰-PE进行初速度测量，结果显示非米氏动力学，具有高低亲和力位点的双相动力学，表明每种[14C]-FFA具有特异性正协同作用。与FFA底物不同，对于两种不同分子种类的PE，即(18:1,18:1)-PE和(16:0,18:2)-PE，未观察到动力学差异，且双相曲线不明显。[14C]-二棕榈酰磷脂酰胆碱和[14C]-棕榈酰辅酶A均不作为合成NAPE的酰基供体，表明其偏好FFA作为酰基供体。此外，乙醇胺和鞘氨醇均未分别作为酰基受体分子形成N-酰基乙醇胺或神经酰胺，表明其对磷脂PE具有特异性。二异丙基氟磷酸（DFP）通过明显修饰一个丝氨酸残基，以时间和浓度依赖性方式使NAPE合酶失活。棕榈酸可保护该酶免受DFP失活和[14C]-DFP掺入，表明一个丝氨酸残基可能在酶的活性位点结合FFA形成酰基-酶中间体。总体而言，这些结果提供了关于一种纯化的整合膜酶动力学行为的新信息，该酶可从两种脂溶性底物合成一种稳定双层的产物。棉籽NAPE合酶的生化特性与体内可能的游离脂肪酸清除作用一致。(c)1998爱思唯尔科学出版社B.V.
