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首页> 外文期刊>Biochemistry >Fluorescence Correlation Spectroscopy of Phosphatidylinositol-Specific Phospholipase C Monitors the Interplay of Substrate and Activator Lipid Binding
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Fluorescence Correlation Spectroscopy of Phosphatidylinositol-Specific Phospholipase C Monitors the Interplay of Substrate and Activator Lipid Binding

机译：磷脂酰肌醇特异性磷脂酶C的荧光相关光谱监测底物和活化剂脂质结合的相互作用

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摘要

Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes simultaneously interact with thensubstrate, PI, and with nonsubstrate lipids such as phosphatidylcholine (PC). For Bacillus thuringiensisnPI-PLC these interactions are synergistic with maximal catalytic activity observed at low to moderate molenfractions of PC (XPC) and maximal binding occurring at low mole fractions of anionic lipids. It has beennproposed that residues in R-helix B help to modulate membrane binding and that dimerization on thenmembrane surface both increases affinity for PC and activates PI-PLC, yielding the observed PI/PC synergy.nVesicle binding and activity measurements using a variety of PI-PLC mutants support many aspects of thisnmodel and reveal that while single mutations can disrupt anionic lipid binding and the anionic lipid/PCnsynergy, the residues important for PC binding are less localized. Interestingly, at high XPC mutations cannboth decrease membrane affinity and increase activity, supporting a model where reductions in wild-typenactivity at XPC>0.6 result from both dilution of the substrate and tight membrane binding of PI-PLC, limitingnenzyme hopping or scooting to the next substrate molecule. These results provide a direct analysis of vesiclenbinding and catalytic activity and shed light on how occupation of the activator site enhances enzymaticnactivity.
机译：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nPI-PLC，这些相互作用与在低至中等摩尔分数的PC（XPC）时观察到的最大催化活性和在低摩尔分数的阴离子脂质中观察到的最大结合是协同的。已经提出，R-螺旋B中的残基有助于调节膜结合，并且在膜表面上的二聚化既增加了对PC的亲和力并激活了PI-PLC，产生了所观察到的PI / PC协同作用。使用多种PI测量囊泡结合和活性-PLC突变体支持该模型的许多方面，并揭示了尽管单个突变可破坏阴离子脂质结合和阴离子脂质/ PCns协同作用，但对PC结合重要的残基较少被定位。有趣的是，在高XPC突变下，既可以降低膜亲和力，又可以增加活性，从而支持一种模型，其中XPC> 0.6时野生型活性降低是由于底物稀释和PI-PLC紧密的膜结合而造成的，从而限制了酶的跳跃或踩踏。底物分子。这些结果提供了对囊泡结合和催化活性的直接分析，并阐明了活化剂位点的占领如何增强酶活性。

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  • 来源
    《Biochemistry》 |2009年第29期|p.6835-6845|共11页
  • 作者

    Mingming Pu Mary F. Roberts and Anne Gershenson;

  • 作者单位

    ‡Department of Chemistry, Boston College, Boston, Massachusetts 02467, and §Department of Chemistry, Brandeis University,Waltham, Massachusetts 02454;

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