Harte R A, Yeaman S J, McElhinney J, Suckling C J, Jackson B, Suckling K E
Department of Biochemistry and Genetics, Medical School, University of Newcastle upon Tyne, UK.
Chem Phys Lipids. 1996 Sep 16;83(1):45-59. doi: 10.1016/0009-3084(96)02593-5.
A series of novel sterols was synthesised as probes for the enzymatic and cellular functions of two important enzymes of intracellular cholesterol metabolism, acyl-CoA:cholesterol acyltransferase (ACAT) and cholesterol 7 alpha-hydroxylase. The compounds were 6-fluoro-5-cholesten-3 beta-ol (6-fluorocholesterol), 7-cholesten-3 beta-ol (7-cholestenol), 6 beta-fluorocholestan-3 beta-ol (6 beta-fluorocholestanol), 3-acetoxy-6-fluorocholestan-3 beta-ol (3-acetoxy-6-fluorocholestanol) and 7-methoxy-5-cholesten-3 beta-ol (7-methoxycholesterol). They were designed to reveal the effect of small changes in sterol structure, particularly reactivity of certain parts of the ring structure and polarity, on enzyme activity and intracellular cholesterol metabolism. The 3 beta-hydroxy group was essential for interaction with both enzymes since 3-acetoxy-6-fluorocholestanol did not affect any of the enzyme-catalysed reactions. 6-Fluorocholesterol and 7-cholestenol had no effect on cholesterol esterification but did inhibit the hydroxylation of cholesterol, as did the other compounds with groups that could influence the 7 position, namely 6 beta-fluorocholestanol and 7-methoxycholesterol. The fluorocholestanols were all competitive substrates for cholesterol esterification in cell-free and cellular assays of ACAT activity. 7-Methoxycholesterol was a surprisingly effective inhibitor of ACAT for a simple sterol. However, 6-fluorocholesterol did not have any effect on ACAT, suggesting that interactions between the enzyme and the region around C-6 and C-7 of the sterol are important. These results show that it is possible to dissect components of cholesterol metabolism using simple, specifically substituted sterols and thus define a new approach to studying the relationships between the various enzymes that catalyse intracellular cholesterol metabolism.
合成了一系列新型甾醇，作为细胞内胆固醇代谢的两种重要酶——酰基辅酶A：胆固醇酰基转移酶（ACAT）和胆固醇7α-羟化酶的酶促功能及细胞功能的探针。这些化合物包括6-氟-5-胆甾烯-3β-醇（6-氟胆固醇）、7-胆甾烯-3β-醇（7-胆甾烯醇）、6β-氟胆甾烷-3β-醇（6β-氟胆甾烷醇）、3-乙酰氧基-6-氟胆甾烷-3β-醇（3-乙酰氧基-6-氟胆甾烷醇）和7-甲氧基-5-胆甾烯-3β-醇（7-甲氧基胆固醇）。它们旨在揭示甾醇结构的微小变化，特别是环结构某些部分的反应性和极性对酶活性及细胞内胆固醇代谢的影响。3β-羟基对于与这两种酶的相互作用至关重要，因为3-乙酰氧基-6-氟胆甾烷醇不影响任何酶催化反应。6-氟胆固醇和7-胆甾烯醇对胆固醇酯化没有影响，但确实抑制了胆固醇的羟基化，其他带有可影响7位基团的化合物，即6β-氟胆甾烷醇和7-甲氧基胆固醇也是如此。在无细胞和细胞ACAT活性测定中，氟胆甾烷醇都是胆固醇酯化的竞争性底物。7-甲氧基胆固醇作为一种简单的甾醇，是一种出人意料的有效ACAT抑制剂。然而，6-氟胆固醇对ACAT没有任何影响，这表明该酶与甾醇C-6和C-7周围区域之间的相互作用很重要。这些结果表明，使用简单的、经过特定取代的甾醇剖析胆固醇代谢的组成部分是可能的，从而确定了一种研究催化细胞内胆固醇代谢的各种酶之间关系的新方法。
