Franklin Christopher L, Li Hui, Martin Stephen F
Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, Texas 78712, USA.
J Org Chem. 2003 Sep 19;68(19):7298-307. doi: 10.1021/jo034411k.
The rate of hydrolysis of natural phospholipids by the phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLC(Bc)) follows the order phosphatidylcholine > phosphatidylethanolamine >> phosphatidyl-l-serine. To probe the structural basis for this substrate specificity, a series of water-soluble, nonhydrolyzable substrate analogues were needed so their complexes with the enzyme could be studied via X-ray crystallography and isothermal titration calorimetry (ITC). Accordingly the water-soluble dithiophospholipids 2-10 having choline, ethanolamine, and l-serine headgroups were synthesized, and the inhibitory activity of each was determined in an assay using 1,2-dihexanoyl-sn-glycero-3-phosphocholine (C6PC) as the monomeric substrate. The 1,2-dibutanoyl dithiophosphocholine 2 was a weak inhibitor, whereas the related 1,2-dipentanoyl dithiophosphocholine 3 and the ethylene glycol dithiophosphocholines 4 and 5 were moderate inhibitors. The 1,2-omega-hydroxydiacyl dithiophosphocholines 6 and 7 were potent inhibitors, while the related compound 8, which had shorter acyl side chains, was a weak inhibitor. The dithiophosphoethanolamine 9 was a modest inhibitor, whereas the dithiophospho-l-serine 10 was a somewhat weaker inhibitor. Overall, the phospholipid analogues had increasing K(i) values according to the order 2 << 10 < 3 < 4 approximately 5 approximately 8 < 9 << 6 << 7 and increasing solubility according to the sequence 5 approximately 7 < 4 approximately 6 approximately 9 < 3 < 10 < 8 < 2.
蜡样芽孢杆菌中偏好磷脂酰胆碱的磷脂酶C（PLC(Bc)）对天然磷脂的水解速率顺序为：磷脂酰胆碱＞磷脂酰乙醇胺＞＞L-丝氨酸磷脂酰。为探究这种底物特异性的结构基础，需要一系列水溶性、不可水解的底物类似物，以便通过X射线晶体学和等温滴定量热法（ITC）研究它们与该酶的复合物。因此，合成了具有胆碱、乙醇胺和L-丝氨酸头部基团的水溶性二硫代磷脂2 - 10，并在以1,2 - 二己酰 - sn - 甘油 - 3 - 磷酸胆碱（C6PC）作为单体底物的测定中确定了每种物质的抑制活性。1,2 - 二丁酰二硫代磷酸胆碱2是一种弱抑制剂，而相关的1,2 - 二戊酰二硫代磷酸胆碱3以及乙二醇二硫代磷酸胆碱4和5是中度抑制剂。1,2 - ω - 羟基二酰基二硫代磷酸胆碱6和7是强效抑制剂，而具有较短酰基侧链的相关化合物8是弱抑制剂。二硫代磷酸乙醇胺9是一种适度抑制剂，而二硫代磷酸 - L - 丝氨酸10是一种稍弱的抑制剂。总体而言，磷脂类似物的抑制常数（Ki）值按2 ＜＜ 10 ＜ 3 ＜ 4 ≈ 5 ≈ 8 ＜ 9 ＜＜ 6 ＜＜ 7的顺序增加，溶解度按5 ≈ 7 ＜ 4 ≈ 6 ≈ 9 ＜ 3 ＜ 10 ＜ 8 ＜ 2的顺序增加。
