Zhou C, Qian X, Roberts M F
Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167, USA.
Biochemistry. 1997 Aug 19;36(33):10089-97. doi: 10.1021/bi970846o.
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 31P 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 diC6PC and diC7PC, 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 diC8PS, 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 31P 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.
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）磷酸酯表现出“界面激活”作用[周等人（1997年），《生物化学》36卷，347 - 355页]。PI-PLC酶在磷脂酰胆碱（PC）或磷脂酰乙醇胺（PE）界面的激活效果最佳。进行了核磁共振实验（TRNOE和³¹P线宽分析）以研究PI-PLC与激活剂两亲分子的相互作用。这些研究表明，该酶对磷脂酰胆碱（或PE）分子具有高亲和力，对二己酰磷脂酰胆碱（diC6PC）和二庚酰磷脂酰胆碱（diC7PC）的解离常数分别为0.5 mM和0.3 mM。使用部分氘代的PC分子证实，结合态PC的TRNOE交叉峰代表分子内弛豫途径，这与单个分子紧密结合一致。PC界面的大幅激活作用可以通过单个PC分子特异性结合到PI-PLC并将酶 - 脂质复合物锚定在界面上来解释。其他界面，如胶束状的二辛酰磷脂酰丝氨酸（diC8PS），可以激活PI-PLC约2 - 至3倍；然而，通过TRNOE或³¹P NMR线宽测量，这些去污剂的单体对该酶几乎没有亲和力。1,2 - 双[12 - （硫辛酸氧基）十二烷酰基] - sn - 甘油 - 3 - 磷酸胆碱的聚合囊泡产生的3.6倍激活作用（与未聚合的PC囊泡产生的15倍激活作用相比）与其他去污剂的非特异性激活作用相当。这证实了单个PC分子的结合是变构的，并将酶锚定在界面上。根据在X射线晶体结构中观察到强度较弱的关键表面环和螺旋B的稳定性，讨论了界面激活酶的构象。
