Hallahan T W, Croteau R
Institute of Biological Chemistry, Washington State University, Pullman 99164-6340.
Arch Biochem Biophys. 1989 Feb 15;269(1):313-26. doi: 10.1016/0003-9861(89)90113-6.
The conversion of geranyl pyrophosphate to (+)-cis- and (+)-trans-sabinene hydrate by a partially purified cyclase from sweet marjoram (Majorana hortensis) is considered to proceed by the initial ionization and isomerization of the substrate to (-)-(3R)-linalyl pyrophosphate and the subsequent cyclization of this enzyme-bound tertiary allylic intermediate to the monocyclic (+)-(4R)-alpha-terpinyl cation. A 1,2-hydride shift and a second cyclization with water capture of the resulting cation complete the reaction sequence. [6-3H, 14C]Geranyl pyrophosphate, coupled with selective chemical degradation of the resulting sabinene hydrate products, was employed to demonstrate the hydride shift, while separate testing of the linalyl pyrophosphate enantiomers confirmed the involvement of the (3R)-antipode in the cyclization and indicated the cyclization of linalyl pyrophosphate to be faster than the coupled isomerization-cyclization of the geranyl substrate. (1R)- and (1S)-[1-3H, 14C]geranyl pyrophosphates, in conjunction with stereoselective degradations of the biosynthetic products to locate the 3H, were exploited to deduce that configuration at C1 of the substrate was retained in the reaction. These findings suggest the isomerization of the geranyl substrate to be a suprafacial process and the cyclization of the (3R)-linalyl intermediate to proceed via the anti,endo-conformation consistent with the stereo-chemistry of other monoterpene cyclizations and with chemical model studies. Sulfonium ion analogs of the presumptive linalyl and alpha-terpinyl cationic intermediates of the isomerization-cyclization sequence were shown to be potent inhibitors of the enzymatic reaction (Ki = 0.3 and 2.8 microM, respectively), and inhibition was synergized by the presence of inorganic pyrophosphate, indicating that the enzyme recognized and bound more tightly to these ion-paired species than to either cationic or anionic partner alone. Additionally, the enzyme was capable of ionizing (solvolyzing) the noncyclizable substrate analogs 6,7-dihydrogeranyl pyrophosphate and 2,3-methanogeranyl pyrophosphate. These results define the overall stereochemistry of the coupled isomerization-cyclization to sabinene hydrate, demonstrate the 1,2-hydride shift, and confirm the electrophilic nature of this enzymatic reaction type.
甜马郁兰(马郁兰)中部分纯化的环化酶将香叶基焦磷酸转化为(+)-顺式和(+)-反式水合桧烯,据认为该过程首先是底物离子化并异构化为(-)-(3R)-芳樟基焦磷酸,随后这种与酶结合的叔烯丙基中间体环化形成单环(+)-(4R)-α-萜品基阳离子。1,2-氢迁移以及随后生成的阳离子与水的二次环化完成了反应序列。[6-³H,¹⁴C]香叶基焦磷酸,结合对生成的水合桧烯产物进行选择性化学降解,用于证明氢迁移,而对芳樟基焦磷酸对映体的单独测试证实了(3R)-对映体参与环化,并表明芳樟基焦磷酸的环化比香叶基底物的异构化-环化耦合反应更快。(1R)-和(1S)-[1-³H,¹⁴C]香叶基焦磷酸,结合对生物合成产物进行立体选择性降解以定位³H,用于推断底物C1位的构型在反应中得以保留。这些发现表明香叶基底物的异构化是一个同面过程,并且(3R)-芳樟基中间体的环化通过反式、内式构象进行,这与其他单萜环化的立体化学以及化学模型研究一致。异构化-环化序列中假定的芳樟基和α-萜品基阳离子中间体的锍离子类似物被证明是酶促反应的有效抑制剂(Ki分别为0.3和2.8微摩尔),并且无机焦磷酸的存在会增强抑制作用,这表明该酶识别并与这些离子对物种结合得比对单独的阳离子或阴离子伴侣更紧密。此外,该酶能够使不可环化的底物类似物6,7-二氢香叶基焦磷酸和2,3-亚甲基香叶基焦磷酸离子化(溶剂解)。这些结果确定了异构化-环化耦合生成水合桧烯的整体立体化学,证明了1,2-氢迁移,并证实了这种酶促反应类型的亲电性质。