Allosteric activation of phosphatidylinositol-specific phospholipase C: Specific phospholipid binding anchors the enzyme to the interface
Allosteric activation of phosphatidylinositol-specific phospholipase C: Specific phospholipid binding anchors the enzyme to the interface
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DOI：
10.1021/bi970846o
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发表时间：
1997-08-19
期刊：
影响因子：
2.9
通讯作者：
Roberts, MF
中科院分区：
文献类型：
--
作者：
Zhou, C;Qian, XQ;Roberts, MF
来自苏云金芽孢杆菌的磷脂酰肌醇特异性磷脂酶 C (PI-PLC) 对水溶性底物肌醇 1,2-(环)磷酸表现出“界面活化”[Zhou et al, (1997) Biochemistry 36, 347-355]。 PI-PLC 酶的激活通过 PC 或 PE 接口达到最佳效果。进行 NMR 实验（TRNOE 和 P-31 线宽分析）以研究 PI-PLC 与活化剂两亲物的相互作用。这些研究表明，该酶对磷脂酰胆碱（或 PE）分子具有高亲和力，diC(6)PC 和 diC(7)PC 的解离常数分别为 0.5 和 0.3 mM。结合 PC 的 TRNOE 交叉峰被证实代表使用与单分子紧密结合一致的部分全氘化 PC 分子的分子内弛豫途径。 PC 界面的大量激活可以通过与 PI-PLC 特异性结合的单个 PC 分子并将酶-脂质复合物锚定到界面来解释。其他界面，如胶束diC(8)PS，可以激活PI-PLC约2-3倍；然而，根据 TRNOE 或 P-31 NMR 线宽测量，这些去污剂的单体对酶几乎没有亲和力。 1,2-双[12-(硫辛酰氧基)十二烷酰基]-sn-甘油-3-磷酸胆碱聚合囊泡产生的 3.6 倍活化（与非聚合 PC 囊泡产生的 15 倍活化相比）与其他去污剂的非特异性活化相当，这证实了单 PC 分子结合是变构的并将酶锚定在界面中。从 X 射线晶体结构中观察到的弱强度的关键表面环和螺旋 B 的稳定性角度讨论了界面激活酶的构象。
Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis exhibits 'interfacial activation' toward the water-soluble substrate myo-inositol 1,2-(cyclic)phosphate [Zhou et al, (1997) Biochemistry 36, 347-355]. The activation of PI-PLC enzyme is optimal with PC or PE interfaces. NMR experiments (TRNOE and P-31 line width analyses) were carried out to investigate the interaction of PI-PLC with activator amphiphiles. These studies showed that the enzyme had high affinity for phosphatidylcholine (or PE) molecules with dissociation constants of 0.5 and 0.3 mM for diC(6)PC and diC(7)PC, respectively. TRNOE cross-peaks of bound PC were confirmed to represent intramolecular relaxation pathways using partially perdeuterated PC molecules consistent with a single molecule binding tightly. The large activation by a PC interface can be explained by a single PC molecule binding specifically to PI-PLC and anchoring the enzyme-lipid complex to the interface. Other interfaces, such as micellar diC(8)PS, can activate PI-PLC about 2-3-fold; however, the monomers of these detergents showed little affinity for the enzyme as measured by TRNOE or P-31 NMR Line widths. The 3.6-fold activation produced by polymerized vesicles of 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphocholine (compared to the 15-fold activation generated by nonpolymerized PC vesicles) was comparable to the nonspecific activation of other detergents, This confirmed that single-PC molecule binding was allosteric and anchored the enzyme in the interface. The conformation of interfacially activated enzyme is discussed in term of the stabilization of a critical surface loop and helix B observed with weak intensity in the X-ray crystal structure.