Li Lu, Storm Patrik, Karlsson Olof P, Berg Stefan, Wieslander Ake
Department of Biochemistry, Umeå University, Umeå, Sweden.
Biochemistry. 2003 Aug 19;42(32):9677-86. doi: 10.1021/bi034360l.
1,2-Diacylglycerol 3-glucosyltransferase is associated with the membrane surface catalyzing the synthesis of the major nonbilayer-prone lipid alpha-monoglucosyl diacylglycerol (MGlcDAG) from 1,2-DAG in the cell wall-less Acholeplasma laidlawii. Phosphatidylglycerol (PG), but not neutral or zwitterionic lipids, seems to be essential for an active conformation and function of the enzyme. Surface plasmon resonance analysis was employed to study association of the enzyme with lipid bilayers. Binding kinetics could be well fitted only to a two-state model, implying also a (second) conformational step. The enzyme bound less efficiently to liposomes containing only zwitterionic lipids, whereas increasing molar fractions of the anionic PG or cardiolipin (CL) strongly promoted binding by improved association (k(a1)), and especially a decreased rate of return (k(d2)) from the second state. This yielded a very low overall dissociation constant (K(D)), corresponding to an essentially irreversible membrane association. Both liposome binding and consecutive activity of the enzyme correlated with the PG concentration. The importance of the electrostatic interactions with anionic lipids was shown by quenching of both binding and activity with increasing NaCl concentrations, and corroborated in vivo for an active enzyme-green fluorescent protein hybrid in Escherichia coli. Nonbilayer-prone lipids substantially enhanced enzyme-liposome binding by promoting a changed conformation (decreasing k(d2)), similar to the anionic lipids, indicating the importance of hydrophobic interactions and a curvature packing stress. For CL and the nonbilayer lipids, effects on enzyme binding and consecutive activity were not correlated, suggesting a separate lipid control of activity. Similar features were recorded with polylysine (cationic) and polyglutamate (anionic) peptides present, but here probably dependent on the selective charge interactions with the enzyme N- and C-domains, respectively. A lipid-dependent conformational change and PG association of the enzyme were verified by circular dichroism, intrinsic tryptophan, and pyrene-probe fluorescence analyses, respectively. It is concluded that an electrostatic association of the enzyme with the membrane surface is accompanied by hydrophobic interactions and a conformational change. However, specific lipids, the curvature packing stress, and proteins or small molecules bound to the enzyme can modulate the activity of the bound A. laidlawii MGlcDAG synthase.
1,2 - 二酰基甘油3 - 葡萄糖基转移酶与膜表面相关联，在无细胞壁的莱氏无胆甾原体中催化从1,2 - 二酰基甘油合成主要的不易形成非双层结构的脂质α - 单葡萄糖基二酰基甘油（MGlcDAG）。磷脂酰甘油（PG）而非中性或两性离子脂质，似乎对于该酶的活性构象和功能至关重要。采用表面等离子体共振分析来研究该酶与脂质双层的结合。结合动力学仅能很好地拟合到双态模型，这也意味着存在一个（第二个）构象步骤。该酶与仅含两性离子脂质的脂质体结合效率较低，而增加阴离子PG或心磷脂（CL）的摩尔分数通过改善结合（k(a1)），特别是降低从第二状态返回的速率（k(d2)），强烈促进了结合。这产生了非常低的总解离常数（K(D)），对应于基本上不可逆的膜结合。脂质体结合和酶的后续活性均与PG浓度相关。随着NaCl浓度增加，结合和活性均被淬灭，这表明了与阴离子脂质的静电相互作用的重要性，并在体内对大肠杆菌中活性酶 - 绿色荧光蛋白杂交体进行了证实。不易形成非双层结构的脂质通过促进构象改变（降低k(d2)），与阴离子脂质类似，显著增强了酶与脂质体的结合，表明疏水相互作用和曲率堆积应力的重要性。对于CL和非双层脂质，对酶结合和后续活性的影响不相关，表明存在对活性的单独脂质控制。当存在聚赖氨酸（阳离子）和聚谷氨酸（阴离子）肽时记录到了类似特征，但这里可能分别取决于与酶的N端和C端结构域的选择性电荷相互作用。分别通过圆二色性、内在色氨酸和芘探针荧光分析证实了该酶的脂质依赖性构象变化和与PG的结合。得出的结论是，该酶与膜表面的静电结合伴随着疏水相互作用和构象变化。然而，特定的脂质、曲率堆积应力以及与该酶结合的蛋白质或小分子可以调节结合的莱氏无胆甾原体MGlcDAG合酶的活性。
