Optimizing the interfacial binding and activity of a bacterial phosphatidylinositol-specific phospholipase C
Optimizing the interfacial binding and activity of a bacterial phosphatidylinositol-specific phospholipase C
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DOI：
10.1074/jbc.m301207200
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发表时间：
2003-07-04
影响因子：
4.8
通讯作者：
Roberts, MF
中科院分区：
文献类型：
--
作者：
Feng, JW;Bradley, WD;Roberts, MF
来自苏云金芽孢杆菌的磷脂酰肌醇特异性磷脂酶C可以被非底物界面激活，如磷脂酰胆碱胶束或双层。这种激活与两个色氨酸的界面部分插入相对应，即位于螺旋B中的色氨酸47和位于环中的色氨酸242。W47A和W242A都具有较弱的界面结合和较低的动力学界面激活。色氨酸拯救突变，即在W47A突变体的螺旋B的不同位置或W242A突变体的环（残基232-244）重新插入色氨酸，已被用来确定色氨酸在这两种结构特征中的重要性和方向。利用磷酸转移酶和磷酸二酯酶测定以及与磷脂酰胆碱囊泡的结合来评估界面活性所需色氨酸的取向和位置。在螺旋B双突变体中，只有一个突变体I43W/W47A具有与Trp-47相同的色氨酸取向。I43W/W47A显示d -肌醇1,2环磷酸水解的磷脂酰肌醇特异性磷脂酶C （PC）活化恢复。然而，对磷脂酰肌醇的比活性仍然低于野生型酶，对磷脂酰肌醇溶解于30%异丙醇（天然酶的标志）的高活性丧失。在由残基232-244组成的环中的几个位置重新插入色氨酸，部分恢复了PC的激活和酶对脂质界面的亲和力以及异丙醇的激活。G238W/W242A对PC接口的激活和亲和力较野生型增强。这些结果为这种细菌磷脂酰肌醇特异性磷脂酶C如何结合激活PC接口提供了限制。
The phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis can be activated by nonsubstrate interfaces such as phosphatidylcholine micelles or bilayers. This activation corresponds with partial insertion into the interface of two tryptophans, Trp-47 in helix B and Trp-242 in a loop, in the rim of the alphabeta-barrel. Both W47A and W242A have much weaker binding to interfaces and considerably lower kinetic interfacial activation. Tryptophan rescue mutagenesis, reinsertion of a tryptophan at a different place in helix B in the W47A mutant or in the loop (residues 232-244) of the W242A mutant, has been used to determine the importance and orientation of a tryptophan in these two structural features. Phosphotransferase and phosphodiesterase assays, and binding to phosphatidylcholine vesicles were used to assess both orientation and position of tryptophans needed for interfacial activity. Of the helix B double mutants, only one mutant, I43W/W47A, has tryptophan in the same orientation as Trp-47. I43W/W47A shows recovery of phosphatidylinositol-specific phospholipase C (PC) activation of D-myo-inositol 1,2-cyclic phosphate hydrolysis. However, the specific activity toward phosphatidylinositol is still lower than wild type enzyme and high activity with phosphatidylinositol solubilized in 30% isopropyl alcohol ( a hallmark of the native enzyme) is lost. Reinserting a tryptophan at several positions in the loop composed of residues 232-244 partially recovers PC activation and affinity of the enzyme for lipid interfaces as well as activation by isopropyl alcohol. G238W/W242A shows an enhanced activation and affinity for PC interfaces above that of wild type. These results provide constraints on how this bacterial phosphatidylinositol-specific phospholipase C binds to activating PC interfaces.