Lewinsohn E, Gijzen M, Croteau R
Institute of Biological Chemistry, Washington State University, Pullman 99164-6340.
Arch Biochem Biophys. 1992 Feb 14;293(1):167-73. doi: 10.1016/0003-9861(92)90380-f.
The major wound-inducible monoterpene synthase (cyclase) of grand fir (Abies grandis) stems transforms geranyl pyrophosphate to both (-)-alpha-pinene (40%) and (-)-beta-pinene (60%). The enzyme was purified to apparent homogeneity by anion-exchange and hydrophobic interaction chromatography, coupled to discontinuous native polyacrylamide gel electrophoresis at neutral pH and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (also at neutral pH) followed by renaturation in 1% Tween 20 (polyoxyethylenesorbitan monolaurate). The renatured enzyme produced a mixture of isomeric pinenes from geranyl pyrophosphate identical to that generated by the native form. The protein exhibited a molecular weight of 63,000 by gel permeation chromatography and of 62,000 by denaturing gel electrophoresis, indicating that the monomer is active. The enzyme required Mn2+ (Km = 30 microM) for activity, exhibited a Km value of 6 microM for the substrate geranyl pyrophosphate, showed a pH optimum at 7.8 and temperature optimum at 42 degrees C, and was inhibited by pyrophosphate (I50 = 0.17 mM), orthophosphate (I50 = 51 mM), and alpha-pinene, as well as by the histidine-directed reagent diethylpyrocarbonate (I50 = 0.64 mM) and the cysteine-directed reagent p-hydroxymercuribenzoate (I50 = 1.9 microM). Although similar in many respects to constitutive monoterpene cyclases of herbaceous species, this inducible cyclase, the first enzyme of this type to be purified to homogeneity from a conifer, is distinguished by the relatively high pH optimum, and the strict specificity and high affinity for the divalent metal ion cofactor.
大冷杉（Abies grandis）树干中主要的伤口诱导型单萜合酶（环化酶）可将香叶基焦磷酸转化为（-）-α-蒎烯（40%）和（-）-β-蒎烯（60%）。通过阴离子交换和疏水相互作用色谱法，结合在中性pH下的不连续天然聚丙烯酰胺凝胶电泳以及在十二烷基硫酸钠存在下（同样在中性pH）的聚丙烯酰胺凝胶电泳，随后在1%吐温20（聚氧乙烯山梨醇单月桂酸酯）中复性，该酶被纯化至表观均一性。复性后的酶从香叶基焦磷酸产生的异构蒎烯混合物与天然形式产生的相同。通过凝胶渗透色谱法测定该蛋白质的分子量为63,000，通过变性凝胶电泳测定为62,000，表明单体具有活性。该酶活性需要Mn2 +（Km = 30 microM），对底物香叶基焦磷酸的Km值为6 microM，最适pH为7.8，最适温度为42℃，并受到焦磷酸（I50 = 0.17 mM）、正磷酸盐（I50 = 51 mM）、α-蒎烯以及组氨酸导向试剂焦碳酸二乙酯（I50 = 0.64 mM）和半胱氨酸导向试剂对羟基汞苯甲酸（I50 = 1.9 microM）的抑制。尽管在许多方面与草本植物的组成型单萜环化酶相似，但这种诱导型环化酶是从针叶树中纯化至均一性的此类第一种酶，其特点是具有相对较高的最适pH，以及对二价金属离子辅因子具有严格的特异性和高亲和力。
