Cane D E, Pawlak J L, Horak R M
Department of Chemistry, Brown University, Providence, Rhode Island 02912.
Biochemistry. 1990 Jun 12;29(23):5476-90. doi: 10.1021/bi00475a011.
Two enantiomeric analogues of farnesyl pyrophosphate (1) were tested as inhibitors and anomalous substrates of trichodiene synthase, which catalyzes the cyclization of trans,trans-farnesyl pyrophosphate (1) to the sesquiterpene hydrocarbon trichodiene (2). The reaction has been shown to involve preliminary isomerization of 1 to the tertiary allylic isomer nerolidyl pyrophosphate (3) which is cyclized without detectable release of the intermediate from the active site of the cyclase. Both (7S)-trans-6,7-dihydrofarnesyl pyrophosphate (7a) and (7R)-trans-6,7-dihydrofarnesyl pyrophosphate (7b), prepared from (3R)- and (3S)- citronellol (9a and 9b), respectively, proved to be modest competitive inhibitors of trichodiene synthase. The values of Ki(7a), 395 nM, and Ki(7b), 220 nM, were 10-15 times the observed Km for 1 and half the Ki of inorganic pyrophosphate alone. Incubation of either 7a or 7b with trichodiene synthase resulted in formation of a mixture of products which by radio/gas-liquid chromatographic and GC/selected ion mass spectrometric analysis was shown to be composed of 80-85% isomeric trienes 19-21 and 15-20% allylic alcohols 12 and 18. Examination of the water-soluble products resulting from incubation of 7a also revealed the generation of 24% of the isomeric cis-6,7-dihydrofarnesyl pyrophosphate (26). The combined rate of formation of anomalous alcoholic and olefinic products was 10% the Vmax determined for the conversion of 1 to 2. The results can be explained by initial enzyme-catalyzed isomerization of dihydrofarnesyl pyrophosphate (7) to the corresponding tertiary allylic isomer dihydronerolidyl pyrophosphate (8). Since the latter intermediate is unable to cyclize due to the absence of the 6,7-double bond, ionization of 8 and quenching of the resulting ion pair by deprotonation, capture of water, or collapse to the isomeric primary pyrophosphate esters will generate the observed spectrum of anomalous products.
法尼基焦磷酸（1）的两种对映体类似物被用作抗真菌烯合酶的抑制剂和异常底物，该酶催化反式、反式 - 法尼基焦磷酸（1）环化生成倍半萜烃抗真菌烯（2）。已表明该反应涉及1初步异构化为叔烯丙基异构体橙花叔醇焦磷酸（3），其在环化酶活性位点环化时未检测到中间体释放。分别由（3R） - 和（3S） - 香茅醇（9a和9b）制备的（7S） - 反式 - 6,7 - 二氢法尼基焦磷酸（7a）和（7R） - 反式 - 6,7 - 二氢法尼基焦磷酸（7b）被证明是抗真菌烯合酶的适度竞争性抑制剂。Ki（7a）的值为395 nM，Ki（7b）的值为220 nM，是观察到的1的Km值的10 - 15倍，是单独无机焦磷酸Ki值的一半。将7a或7b与抗真菌烯合酶一起孵育会导致产物混合物的形成，通过放射性/气液色谱和GC /选择离子质谱分析表明该混合物由80 - 85％的异构三烯19 - 21和15 - 20％的烯丙醇12和18组成。对7a孵育产生的水溶性产物的检查还揭示了24％的异构顺式 - 6,7 - 二氢法尼基焦磷酸（26）的生成。异常醇类和烯烃类产物的总生成速率是1转化为2所确定的Vmax的10％。这些结果可以通过二氢法尼基焦磷酸（7）最初的酶催化异构化为相应的叔烯丙基异构体二氢橙花叔醇焦磷酸（8）来解释。由于后一种中间体由于没有6,7 - 双键而无法环化，8的电离以及通过去质子化、捕获水或塌缩为异构伯焦磷酸酯来淬灭所得离子对将产生观察到的异常产物光谱。
