Laboratory of Molecular Neurobiology and Biophysics, The Rockefeller University, New York, NY 10065.
HHMI, The Rockefeller University, New York, NY 10065.
Proc Natl Acad Sci U S A. 2023 May 16;120(20):e2301121120. doi: 10.1073/pnas.2301121120. Epub 2023 May 12.
catalyze the hydrolysis of phosphatidylinositol 4, 5-bisphosphate [Formula: see text] into [Formula: see text] [Formula: see text] and [Formula: see text] [Formula: see text]. [Formula: see text] regulates the activity of many membrane proteins, while and lead to increased intracellular Ca levels and activate protein kinase C, respectively. are regulated by G protein-coupled receptors through direct interaction with [Formula: see text] and [Formula: see text] and are aqueous-soluble enzymes that must bind to the cell membrane to act on their lipid substrate. This study addresses the mechanism by which [Formula: see text] activates 3. We show that 3 functions as a slow Michaelis-Menten enzyme ( [Formula: see text] ) on membrane surfaces. We used membrane partitioning experiments to study the solution-membrane localization equilibrium of 3. Its partition coefficient is such that only a small quantity of 3 exists in the membrane in the absence of [Formula: see text] . When [Formula: see text] is present, equilibrium binding on the membrane surface increases 3 in the membrane, increasing [Formula: see text] in proportion. Atomic structures on membrane vesicle surfaces show that two [Formula: see text] anchor 3 with its catalytic site oriented toward the membrane surface. Taken together, the enzyme kinetic, membrane partitioning, and structural data show that [Formula: see text] activates by increasing its concentration on the membrane surface and orienting its catalytic core to engage [Formula: see text] . This principle of activation explains rapid stimulated catalysis with low background activity, which is essential to the biological processes mediated by [Formula: see text], and .
催化磷脂酰肌醇 4,5-二磷酸[公式：见文本]水解为[公式：见文本] [公式：见文本]和[公式：见文本]。[公式：见文本]调节许多膜蛋白的活性，而[公式：见文本]和[公式：见文本]导致细胞内 Ca 水平升高，分别激活蛋白激酶 C。通过与[公式：见文本]和[公式：见文本]直接相互作用，[公式：见文本]受 G 蛋白偶联受体调节，并且是水溶性酶，必须与细胞膜结合才能作用于其脂质底物。本研究探讨了[公式：见文本]激活 3 的机制。我们表明，3 在膜表面上作为缓慢的米氏酶（[公式：见文本]）起作用。我们使用膜分区实验研究 3 在溶液-膜定位平衡中的作用。其分配系数使得在没有[公式：见文本]的情况下，只有少量的 3 存在于膜中。当[公式：见文本]存在时，膜表面上的平衡结合增加了膜中的 3，相应地增加了[公式：见文本]。膜囊泡表面的原子结构表明，两个[公式：见文本]将 3 锚定在膜上，其催化位点朝向膜表面。综上所述，酶动力学、膜分区和结构数据表明，[公式：见文本]通过增加其在膜表面上的浓度并将其催化核心定向与[公式：见文本]结合来激活 3。这种激活原理解释了具有低背景活性的快速刺激催化，这对于由[公式：见文本]和[公式：见文本]介导的生物过程至关重要。
