Fluorescence Correlation Spectroscopy of Phosphatidylinositol-Specific Phospholipase C Monitors the Interplay of Substrate and Activator Lipid Binding
Fluorescence Correlation Spectroscopy of Phosphatidylinositol-Specific Phospholipase C Monitors the Interplay of Substrate and Activator Lipid Binding
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DOI：
10.1021/bi900633p
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发表时间：
2009-07-28
期刊：
影响因子：
2.9
通讯作者：
Gershenson, Anne
中科院分区：
文献类型：
--
作者：
Pu, Mingming;Roberts, Mary F.;Gershenson, Anne
磷脂酰肌醇特异性磷脂酶C（PI-PLC）酶同时与底物PI和非底物脂质（如磷脂酰胆碱（PC））相互作用。对于苏云金芽孢杆菌PI-PLC，这些相互作用是协同的，在低至中等摩尔分数的PC（X-PC）和最大结合发生在低摩尔分数的阴离子脂质的最大催化活性。已经提出α-螺旋B中的残基有助于调节膜结合，并且膜表面的二聚化油既增加了对PC的亲和力又激活了PI-PLC，从而产生了观察到的PI/PC协同作用。使用各种PI-PLC突变体的囊泡结合和活性测量支持该模型的许多方面，并揭示虽然单个突变破坏阴离子脂质结合和阴离子脂质/PC协同作用，但对PC结合重要的残基较少定位。有趣的是，在高X-PC下，突变可以降低膜亲和力并增加活性，支持这样的模型，其中在X-PC > 0.6下野生型活性的降低是由于底物的稀释和PI-PLC的紧密膜结合，限制酶跳跃或快速移动到下一个底物分子。这些结果提供了一个直接的囊泡结合和催化活性的分析，并阐明如何占领的激活位点增强酶的活性。
Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes simultaneously interact with the substrate, PI, and with nonsubstrate lipids such as phosphatidylcholine (PC). For Bacillus thuringiensis PI-PLC these interactions are synergistic with maximal catalytic activity observed at low to moderate mole fractions of PC (X-PC) and maximal binding occurring at low mole fractions of anionic lipids. It has been proposed that residues in alpha-helix B help to modulate membrane binding and that dimerization oil the membrane surface both increases affinity for PC and activates PI-PLC, yielding the observed PI/PC synergy. Vesicle binding and activity measurements using a variety of PI-PLC Mutants support many aspects of this model and reveal that while single mutations call disrupt anionic lipid binding and the anionic lipid/PC synergy, the residues important for PC binding are less localized. Interestingly, at high X-PC mutations can both decrease membrane affinity and increase activity, supporting a model where reductions in wild-type activity at X-PC > 0.6 result from both dilution of the substrate and tight membrane binding of PI-PLC, limiting enzyme hopping or scooting to the next substrate molecule. These results provide a direct analysis of vesicle binding and catalytic activity and shed light on how occupation of the activator site enhances enzymatic activity.