Wiedemann M, Misawa N, Sandmann G
Lehrstuhl für Physiologie und Biochemie der Pflanzen, Universität Konstanz, Germany.
Arch Biochem Biophys. 1993 Oct;306(1):152-7. doi: 10.1006/abbi.1993.1493.
Geranylgeranyl pyrophosphate (GGPP) synthase from Erwinia uredovora was overexpressed in Escherichia coli and purified to homogeneity from solubilized inclusion bodies. In this protein the first 13 N-terminal amino acids were replaced by 16 other amino acids resulting from the cloning vector pUC18. Nevertheless, the enzyme showed activity after purification which could be stimulated sixfold by appropriate activation conditions. The homogeneous enzyme was used to study substrate and product specificity as well as to determine Km values for isopentenyl pyrophosphate, dimethylallyl pyrophosphate (DMAPP), geranyl pyrophosphate (GPP), and farnesyl pyrophosphate (FPP). Reaction rates and Km values indicate that FPP and GPP are the genuine allylic substrates for GGPP synthase, but not DMAPP. Independent of the allylic substrate employed, GGPP was the only reaction product of the enzymatic reaction.
来自丁香假单胞菌的香叶基香叶基焦磷酸合酶（GGPP）在大肠杆菌中过表达，并从溶解的包涵体中纯化至同质。在该蛋白质中，前13个N端氨基酸被克隆载体pUC18产生的其他16个氨基酸取代。然而，该酶在纯化后仍显示出活性，通过适当的激活条件可被刺激6倍。使用该同质酶研究底物和产物特异性，并确定异戊烯基焦磷酸、二甲基烯丙基焦磷酸（DMAPP）、香叶基焦磷酸（GPP）和法尼基焦磷酸（FPP）的Km值。反应速率和Km值表明FPP和GPP是GGPP合酶真正的烯丙基底物，而不是DMAPP。无论使用何种烯丙基底物，GGPP都是酶促反应的唯一反应产物。
