Interfacial kinetic analysis of the tumour suppressor phosphatase, PTEN: evidence for activation by anionic phospholipids
Interfacial kinetic analysis of the tumour suppressor phosphatase, PTEN: evidence for activation by anionic phospholipids
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DOI：
10.1042/bj20021848
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发表时间：
2003-05-01
影响因子：
4.1
通讯作者：
Downes, CP
中科院分区：
文献类型：
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作者：
McConnachie, G;Pass, I;Downes, CP
我们研究了动力学行为和底物特异性的PTEN（磷酸酶和张力蛋白同源10号染色体上删除）使用单层囊泡含有底物脂质的背景磷脂酰胆碱。PTEN显示界面酶的预期特征，因为酶活性的速率取决于所用底物脂质的表面浓度（摩尔分数）以及本体浓度。表面稀释分析显示，PTEN对PtdIns（3，4，5）P-3的催化效率比PtdIns（3，4）P-2或PtdIns（3，5）P-2高200倍，比PtdIns 3 P高1000倍。界面K. PtdIns（3，4，5）P-3的PTEN值非常低，反映了存在于细胞膜中的这种脂质的小比例。PtdIns（3，4，5）P-3的催化中心活性（k（cat））至少是水溶性底物Ins（1，3，4，5）P-4的200倍。对脂质底物的偏好可能是由于酶的界面活化，而不是囊泡底物的过程催化。此外，PtdIns（4，5）P-2和单价盐均刺激PTEN对PtdIns（3，4，5）P-3的活性，但显著抑制对Ins（1，3，4，5）P-4的活性。PtdIns（4，5）P-2的刺激作用与其他阴离子磷脂物质相比在幅度上更大且更有效。在该模型中，无生物活性的脂质结合C2结构域（M-CBR 3）中的突变没有改变整体催化效率，但降低了界面结合步骤的效率，证明了其在PTEN催化机制中的重要性。
We investigated the kinetic behaviour and substrate specificity of PTEN (phosphatase and tensin homologue deleted on chromosome 10) using unilamellar vesicles containing substrate lipids in a background of phosphatidylcholine. PTEN displays the characteristics expected of an interfacial enzyme, since the rate of enzyme activity is dependent on the surface concentration of the substrate lipids used (mol fraction), as well as the bulk concentration. Surface-dilution analysis revealed the catalytic efficiency of PTEN for PtdIns(3,4,5)P-3 to be 200-fold greater than for either PtdIns(3,4)P-2 or PtdIns(3,5)P-2, and 1000-fold greater than for PtdIns3P. The interfacial K. value of PTEN for PtdIns(3,4,5)P-3 was very low, reflecting the small proportions of this lipid that are present in cellular membranes. The catalytic-centre activity (k(cat)) for PtdIns(3,4,5)P-3 was at least 200-fold greater than that for the water-soluble substrate Ins(1,3,4,5)P-4. The preference for lipid substrates may result from an interfacial activation of the enzyme, rather than processive catalysis of vesicular substrates. Moreover, both PtdIns(4,5)P-2 and univalent salts stimulated the activity of PTEN for PtdIns(3,4,5)P-3, but profoundly inhibited activity against Ins(1,3,4,5)P-4. The stimulatory effect of PtdIns(4,5)P-2 was greater in magnitude and more potent in comparison with other anionic phospholipid species. A mutation in the lipid-binding C2 domain (M-CBR3) that is biologically inactive did not alter overall catalytic efficiency in this model, but decreased the efficiency of the interfacial binding step, demonstrating its importance in the catalytic mechanism of PTEN.