Activation of phosphatidylinositol-specific phospholipase C toward inositol 1,2-(cyclic)-phosphate.
Activation of phosphatidylinositol-specific phospholipase C toward inositol 1,2-(cyclic)-phosphate.
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磷脂酰肌醇特异性磷脂酶 C 向肌醇 1,2-（环）-磷酸的激活。
DOI：
10.1021/bi960601w
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Roberts,MF
中科院分区：
文献类型：
--
作者：
Zhou,C;Wu,Y;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) 通过离散步骤催化磷脂酰肌醇 (PI) 的水解： (i) 分子内磷酸转移酶反应形成肌醇 1,2-(环状)-磷酸 (cIP)，然后是 (ii) 将 cIP 转化为肌醇 1-磷酸的环状磷酸二酯酶活性。以水溶性cIP为底物，研究PI-PLC的环状磷酸二酯酶活性和界面行为。使用不同的去污剂胶束和磷脂囊泡来检查酶是否可以针对可溶性底物发生“界面活化”。几乎所有检查的去垢剂都将酶激活至少 2 倍，其中 PC 种类使 PI-PLC 比活性增加最大。在几种代表性去污剂（例如 Triton X-100 和二庚酰磷脂酰胆碱 (diC7PC)）存在和不存在的情况下测量动力学参数。凝胶过滤实验表明，在这些条件下，cIP 不会与这些去污剂胶束发生任何可测量的分配。观察到最大活化一半的浓度发生在去污剂 CMC 附近。 Km 和 Vmax 都因表面的存在而改变：根据洗涤剂的不同，Km 会不同程度地降低，而 Vmax 则大幅增加。没有去垢剂时，cIP 的 Km 为 90 mM，添加 diC7PC 胶束后降至 29 mM；存在 diC7PC 胶束时，Vmax 增加了近 7 倍。 diC7PC 存在下，酶效率 (Vmax/Km) 增加了 21 倍以上，但仍比单体二己酰磷脂酰肌醇的初始磷酸转移酶活性低 20 倍。环磷酸二酯酶活性的低效率很大程度上是由于底物结合亲和力造成的。速率对底物浓度的依赖性表现出协同行为，尤其是在没有洗涤剂的情况下。这种协同性是根据酶上的蛋白质聚集和配体结合位点来讨论的。
Phosphatidylinositol-specific phospholipase C (PI-PLC) fromBacillus thuringiensiscatalyzes the hydrolysis of phosphatidylinositol (PI) in discrete steps: (i) an intramolecular phosphotransferase reaction to form inositol 1,2-(cyclic)-phosphate (cIP), followed by (ii) a cyclic phosphodiesterase activity that converts cIP to inositol 1-phosphate. Water-soluble cIP was used as the substrate to study the cyclic phosphodiesterase activity and interfacial behavior of PI-PLC. Different detergent micelles and phospholipid vesicles were used to examine if “interfacial activation” of the enzyme could occur toward a soluble substrate. Almost all detergents examined activated the enzyme at least 2-fold, with PC species yielding the largest increases in PI-PLC specific activity. Kinetic parameters were measured in the absence and presence of several representative detergents (e.g., Triton X-100 and diheptanoylphosphatidylcholine (diC7PC)). Gel filtration experiments showed that, under these conditions, the cIP did not partition to any measurable extent with these detergent micelles. The concentration at which half the maximum activation was observed occurred near the detergent CMC. BothKmandVmaxwere altered by the presence of a surface:Kmdecreased to different degrees depending on the detergent, whileVmaxincreased substantially. TheKmfor cIP was 90 mM without detergent and decreased to 29 mM with diC7PC micelles added;Vmaxincreased almost 7-fold in the presence of diC7PC micelles. The enzyme efficiency (Vmax/Km) in the presence of diC7PC increased more than 21-fold, but it was still 20-fold lower than initial phosphotransferase activity for monomeric dihexanoylphosphatidylinositol. The poor efficiency of the cyclic phosphodiesterase activity is largely due to substrate binding affinity. The dependence of rate on substrate concentration exhibits cooperative behavior, especially without detergent. This cooperativity is discussed in terms of protein aggregation and ligand binding sites on the enzyme.