Croteau R, Wheeler C J, Aksela R, Oehlschlager A C
J Biol Chem. 1986 Jun 5;261(16):7257-63.
The enzymatic cyclization of geranyl pyrophosphate to monoterpenes is thought to proceed through a series of carbocation-pyrophosphate anion paired intermediates. Sulfonium analogs of two putative carbocationic intermediates of the cyclization sequence were shown to be inhibitors of the conversion of the acyclic precursor to the bicyclic monoterpenes (+)-alpha-pinene and (+)-bornyl pyrophosphate by partially purified cyclase preparations from sage (Salvia officinalis). The sulfonium analog of the tertiary allylic, linalyl, intermediate (i.e. methyl-(4-methylpent-3-en-1-yl)vinyl-sulfonium perchlorate) provided respective Ki values of 2.5 microM and 3.0 microM against the cyclization to alpha-pinene and bornyl pyrophosphate at a substrate concentration of 5 microM, whereas the sulfonium analog of the monocyclic, alpha-terpinyl, intermediate (i.e. dimethyl-(4-methylcyclohex-3-en-1-yl) sulfonium iodide) exhibited respective Ki values of 3.4 microM and 3.9 microM against the same two cyclizations. The potency of inhibition in all cases increased with increasing substrate concentration, indicating that the affinity of the enzymes for the sulfonium analogs was increased by the presence of the pyrophosphate ester. Inorganic pyrophosphate at a concentration of 50 microM, which alone had little influence on the cyclizations, increased the effectiveness of inhibition of the sulfonium analogs severalfold, and the apparent Ki for inorganic pyrophosphate was reduced manyfold by the presence of either analog at 5 microM. That the combination of sulfonium analog and pyrophosphate provided synergistic inhibition of the electrophilic cyclizations indicated that the cyclases bind the paired species more tightly than either partner alone. Specificity studies suggested that inhibition by the above sulfonium ion:pyrophosphate pairs was due to both electronic and structural resemblance to intermediates of the reaction.
香叶基焦磷酸酶促环化生成单萜的过程被认为是通过一系列碳正离子 - 焦磷酸根阴离子配对中间体进行的。环化序列中两个假定的碳正离子中间体的锍类似物被证明是鼠尾草（Salvia officinalis）部分纯化的环化酶制剂将无环前体转化为双环单萜（+）-α-蒎烯和（+）-冰片基焦磷酸的抑制剂。叔烯丙基、芳樟醇中间体的锍类似物（即甲基 - （4 - 甲基戊 - 3 - 烯 - 1 - 基）乙烯基高氯酸锍）在底物浓度为5μM时，对环化生成α-蒎烯和冰片基焦磷酸的抑制常数（Ki）分别为2.5μM和3.0μM，而单环、α-萜品基中间体的锍类似物（即二甲基 - （4 - 甲基环己 - 3 - 烯 - 1 - 基）碘化锍）对相同的两种环化反应的Ki值分别为3.4μM和3.9μM。在所有情况下，抑制效力都随底物浓度增加而增强，这表明焦磷酸酯的存在增加了酶对锍类似物的亲和力。浓度为50μM的无机焦磷酸单独对环化反应影响很小，但能使锍类似物的抑制效果增强几倍，并且5μM的任何一种类似物的存在都会使无机焦磷酸的表观Ki值降低很多倍。锍类似物和焦磷酸的组合对亲电环化反应具有协同抑制作用，这表明环化酶结合配对物质的紧密程度高于单独结合任何一种成分。特异性研究表明，上述锍离子：焦磷酸对的抑制作用是由于与反应中间体在电子和结构上的相似性。
