Han S K, Yoon E T, Scott D L, Sigler P B, Cho W
Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois 60607-7061, USA.
J Biol Chem. 1997 Feb 7;272(6):3573-82.
Recent genetic and structural studies have shed considerable light on the mechanism by which secretory phospholipases A2 interact with substrate aggregates. Electrostatic forces play an essential role in optimizing interfacial catalysis. Efficient and productive adsorption of the Class I bovine pancreatic phospholipase A2 to anionic interfaces is dependent upon the presence of two nonconserved lysine residues at sequence positions 56 and 116, implying that critical components of the adsorption surface differ among enzyme species (Dua, R., Wu, S.-K., and Cho, W. (1995) J. Biol. Chem. 270, 263-268). In an effort to further characterize the protein residues involved in interfacial catalysis, we have determined the high resolution (1.7 A) x-ray structure of the Class II Asp-49 phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus. Correlation of the three-dimensional coordinates with kinetic data derived from site-directed mutations near the amino terminus (E6R, K7E, K10E, K11E, and K16E) and the active site (K54E and K69Y) defines much of the interface topography. Lysine residues at sequence positions 7 and 10 mediate the adsorption of A. p. piscivorus phospholipase A2 to anionic interfaces but play little role in the enzyme's interaction with electrically neutral surfaces or in substrate binding. Compared to the native enzyme, the mutant proteins K7E and K10E demonstrate comparable (20-fold) decreases in affinity and catalysis on polymerized mixed liposomes of 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine and 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphoglycerol, while the double mutant, K7E/K10E, shows a more dramatic 500-fold decrease in catalysis and interfacial adsorption. The calculated contributions of Lys-7 and Lys-10 to the free energy of binding of A. p. piscivorus phospholipase A2 to anionic liposomes (-1.8 kcal/mol at 25 degrees C per lysine) are additive (i.e. -3.7 kcal/mol) and together represent nearly half of the total binding energy. Although both lysine side chains lie exposed at the edge of the proposed interfacial adsorption surface, they are geographically remote from the corresponding interfacial determinants for the bovine enzyme. Our results confirm that interfacial adsorption is largely driven by electrostatic forces and demonstrate that the arrangement of the critical charges (e.g. lysines) is species-specific. This variability in the topography of the adsorption surface suggests a corresponding flexibility in the orientation of the active enzyme at the substrate interface.
最近的遗传学和结构研究为分泌型磷脂酶A2与底物聚集体相互作用的机制提供了相当多的见解。静电力在优化界面催化中起着至关重要的作用。I类牛胰磷脂酶A2对阴离子界面的高效且有效的吸附取决于序列位置56和116处两个非保守赖氨酸残基的存在，这意味着吸附表面的关键成分在不同酶种之间存在差异（杜阿，R.，吴，S.-K.，和赵，W.（1995年）《生物化学杂志》270，263 - 268）。为了进一步表征参与界面催化的蛋白质残基，我们测定了来自食鱼蝮蛇毒液的II类天冬氨酸-49磷脂酶A2的高分辨率（1.7 Å）X射线结构。三维坐标与从氨基末端附近（E6R、K7E、K10E、K11E和K16E）以及活性位点（K54E和K69Y）的定点突变获得的动力学数据的相关性定义了大部分界面拓扑结构。序列位置7和10处的赖氨酸残基介导食鱼蝮蛇磷脂酶A2对阴离子界面的吸附，但在该酶与电中性表面的相互作用或底物结合中作用很小。与天然酶相比，突变蛋白K7E和K10E在1 - 十六烷酰基-2 -（1 - 芘癸酰基）-sn -甘油-3 -磷酸胆碱和1,2 -双[12 -（硫辛酸氧基）十二烷酰基]-sn -甘油-3 -磷酸甘油的聚合混合脂质体上的亲和力和催化作用表现出相当的（20倍）下降，而双突变体K7E/K10E在催化和界面吸附方面表现出更显著的500倍下降。计算得出的赖氨酸-7和赖氨酸-10对食鱼蝮蛇磷脂酶A2与阴离子脂质体结合自由能的贡献（在25℃下每个赖氨酸为-1.8千卡/摩尔）是可加的（即-3.7千卡/摩尔），并且一起几乎占总结合能的一半。尽管两个赖氨酸侧链都暴露在所提出的界面吸附表面的边缘，但它们在空间上远离牛酶的相应界面决定因素。我们的结果证实界面吸附在很大程度上由静电力驱动，并表明关键电荷（如赖氨酸）的排列是物种特异性的。吸附表面拓扑结构的这种变异性表明活性酶在底物界面处的取向具有相应的灵活性。
