Chen A, Zhang D, Poulter C D
Department of Chemistry, University of Utah, Salt Lake City 84112.
J Biol Chem. 1993 Oct 15;268(29):21701-5.
The first pathway-specific step in the biosynthesis of the core membrane diether lipids in archaebacteria is the alkylation of the primary hydroxyl group in (S)-glyceryl phosphate by geranylgeranyl diphosphate. The reaction is catalyzed by (S)-3-O-geranylgeranylglyceryl phosphate ((S)-GGGP) synthase. The cytosolic enzyme was purified to homogeneity from the moderately thermophilic archaebacterium Methanobacterium thermoautotrophicum by a combination of ammonium sulfate precipitation, four chromatographic steps (DE52, Q-Sepharose, phenyl-Superose, and Protein Pak), and native polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis of gel-purified GGGP synthase gave a single band at 29 kDa. The enzyme requires Mg2+ for optimal activity, although prenyltransfer is also seen in buffers containing Mn2+ or Zn2+. A well defined pH optimum occurs between 6.0 and 7.5. Maximal activity is seen at 50-65 degrees C. The Michaelis constants for GGGP synthase are Vmax = 4.1 +/- 0.5 mumol min-1 mg-1, KMGGPP = 4.1 +/- 1.1 microM, and KMGP = 41 +/- 5 microM.
古细菌核心膜二醚脂质生物合成过程中第一个特定途径步骤是香叶基香叶基二磷酸对（S）-甘油磷酸中伯羟基的烷基化反应。该反应由（S）-3-O-香叶基香叶基甘油磷酸（（S）-GGGP）合酶催化。通过硫酸铵沉淀、四个色谱步骤（DE52、Q-Sepharose、苯基-Superose和Protein Pak）以及天然聚丙烯酰胺凝胶电泳相结合的方法，从嗜热古细菌嗜热自养甲烷杆菌中纯化出了均一的胞质酶。凝胶纯化的GGGP合酶经SDS-聚丙烯酰胺凝胶电泳后在29 kDa处呈现单一条带。该酶需要Mg2+才能达到最佳活性，不过在含有Mn2+或Zn2+的缓冲液中也能观察到异戊二烯基转移反应。其最适pH在6.0至7.5之间。在50 - 65摄氏度时可观察到最大活性。GGGP合酶的米氏常数分别为：Vmax = 4.1 +/- 0.5 μmol min-1 mg-1，KMGGPP = 4.1 +/- 1.1 μM，以及KMGP = 41 +/- 5 μM。
