Nes W D, Janssen G G, Bergenstrahle A
Plant and Fungal Lipid Research, Richard B. Russell Research Center, Athens, Georgia 30613.
J Biol Chem. 1991 Aug 15;266(23):15202-12.
The membrane-bound enzyme of microsomes obtained from sunflower embryos that catalyzes the bi-substrate transfer reaction whereby the methyl group of (S)-adenosyl-L-methionine is transferred to C-24 of the sterol side chain has been investigated. Optimal incubation conditions for assay of the microsomal (S)-adenosyl-L-methionine:sterol delta 24-methyl transferase (SMT) have been established for the first time. The microsomal preparation was found to catalyze the formation of a delta 24(28)-sterol and to be free of contaminating methyl transferase enzymes, e.g. those which form delta 23-24 methyl sterols (cyclosadol) and delta 25-24 beta-methyl sterols (cyclolaudenol) and other sterolic enzymes which might transform the acceptor molecule to metabolites which could compete in the assay with the test substrate. From a series of incubations with 27 sterol and sterol-like (triterpenoids) substrates of which 23 compounds possessed a 24,25-double bond, we observed a marked dependence on precise structural features and three-dimensional shape of the acceptor molecule in its ability to be transformed by the SMT. In contrast to the yeast SMT where cycloartenol fails to bind to the SMT and zymosterol is the best substrate for methylation, the sunflower SMT studied here utilizes cycloartenol preferentially to zymosterol and the other substrates. Of the chemical groups which distinguishes cycloartenol, a free 3 beta-OH,9 beta,19-cyclopropyl group, trimethylated saturated nucleus, and delta 24-double bond, only the nucleophilic centers at C-3 and C-24 were obligatory for substrate binding and methylation. Of the bent or flat conformations which cycloartenol may orient in the enzyme-substrate complex, our results indicate a selection for acceptor molecules which possess the shape that closely resembles the crystal state and solution orientation of cycloartenol which is now known to be flat rather than bent (Nes, W. D., Benson, M., Lundin, R. E., and Le, P. H. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5759-5763).
对从向日葵胚中获得的微粒体膜结合酶进行了研究，该酶催化双底物转移反应，即将（S）-腺苷-L-甲硫氨酸的甲基转移至甾醇侧链的C-24位。首次确定了微粒体（S）-腺苷-L-甲硫氨酸：甾醇δ24-甲基转移酶（SMT）测定的最佳孵育条件。发现微粒体制剂可催化δ24（28）-甾醇的形成，且不含污染性甲基转移酶，例如那些形成δ23-24甲基甾醇（环阿屯醇）和δ25-24β-甲基甾醇（环劳丹醇）的酶以及其他可能将受体分子转化为可在测定中与测试底物竞争的代谢物的甾醇酶。在与27种甾醇和甾醇类（三萜类）底物的一系列孵育中，其中23种化合物具有24,25-双键，我们观察到受体分子被SMT转化的能力对其精确的结构特征和三维形状有明显依赖性。与酵母SMT不同，在酵母SMT中，环阿屯醇不能与SMT结合，而酵母甾醇是甲基化的最佳底物，此处研究的向日葵SMT优先利用环阿屯醇而非酵母甾醇和其他底物。在区分环阿屯醇的化学基团中，游离的3β-OH、9β,19-环丙基、三甲基化饱和核和δ24-双键，只有C-3和C-24处的亲核中心对于底物结合和甲基化是必需的。在环阿屯醇可能在酶-底物复合物中呈现的弯曲或平面构象中，我们的结果表明选择了具有与环阿屯醇的晶体状态和溶液取向非常相似形状的受体分子，现在已知环阿屯醇是平面的而非弯曲的（内斯，W.D.，本森，M.，伦丁，R.E.，和勒，P.H.（1988年）美国国家科学院院刊85，5759-5763）。
