Alonso W R, Croteau R
Institute of Biological Chemistry, Washington State University, Pullman 99164-6340.
Arch Biochem Biophys. 1991 May 1;286(2):511-7. doi: 10.1016/0003-9861(91)90073-r.
The monoterpene cyclase, gamma-terpinene synthase, from Thymus vulgaris (thyme) leaves was purified to apparent homogeneity by isoelectric focusing and dye-ligand, anion-exchange, hydrophobic interaction, and gel permeation chromatography. The enzyme has a native molecular weight of 96,000 as determined by gel permeation chromatography, and exhibited a specific activity of 538 nmol/h.mg protein (turnover number of approximately 0.01/s). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the enzyme to be composed of two apparently identical subunits of Mr approximately 55,000. The protein was very hydrophobic, and possessed a pI value of 4.85 as determined by isoelectric focusing. Maximum activity was observed at pH 6.8 in the presence of 20 mM Mg2+; 5 mM Mn2+ could support catalysis, albeit at a much lower rate. The Km value for the substrate, geranyl pyrophosphate, was 2.6 microM. Cyclase activity was inhibited by cysteine- and histidine-directed reagents. Purified gamma-terpinene synthase also possessed the ability to cyclize geranyl pyrophosphate to small amounts of alpha-thujene and to lesser quantities of myrcene, alpha-terpinene, limonene, linalool, terpinen-4-ol, and alpha-terpineol, all of which appear to be coproducts of the reaction sequence leading to gamma-terpinene. In general properties, the gamma-terpinene synthase from thyme leaves resembles other monoterpene cyclases as well as sesquiterpene and diterpene cyclases.
通过等电聚焦、染料配体、阴离子交换、疏水相互作用和凝胶渗透色谱法，从百里香叶中纯化出单萜环化酶γ-萜品烯合酶，使其达到表观均一性。通过凝胶渗透色谱法测定，该酶的天然分子量为96,000，比活性为538 nmol/h·mg蛋白（周转数约为0.01/s）。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示该酶由两个表观相同的亚基组成，亚基的分子量约为55,000。该蛋白具有很强的疏水性，通过等电聚焦测定其pI值为4.85。在20 mM Mg2+存在下，pH 6.8时观察到最大活性；5 mM Mn2+也能支持催化作用，尽管速率要低得多。底物香叶基焦磷酸的Km值为2.6 μM。环化酶活性受到半胱氨酸和组氨酸定向试剂的抑制。纯化的γ-萜品烯合酶还具有将香叶基焦磷酸环化生成少量α-侧柏烯和更少量月桂烯、α-萜品烯、柠檬烯、芳樟醇、萜品-4-醇和α-松油醇的能力，所有这些似乎都是导致γ-萜品烯的反应序列的副产物。总体而言，百里香叶中的γ-萜品烯合酶与其他单萜环化酶以及倍半萜和二萜环化酶相似。
