Shepherd Mark, Reid James D, Hunter C Neil
Robert Hill Institute for Photosynthesis, Department of Molecular Biology and Biotechnology, Firth Court, Western Bank, University of Sheffield, Sheffield S10 2TN, UK.
Biochem J. 2003 Apr 15;371(Pt 2):351-60. doi: 10.1042/BJ20021394.
Magnesium protoporphyrin IX methyltransferase (ChlM), catalyses the methylation of magnesium protoporphyrin IX (MgP) at the C(6) propionate side chain to form magnesium protoporphyrin IX monomethylester (MgPME). Threading methods biased by sequence similarity and predicted secondary structure have been used to assign this enzyme to a particular class of S-adenosyl-L-methionine (SAM)-binding proteins. These searches suggest that ChlM contains a seven-stranded beta-sheet, common among small-molecule methyltransferases. Steady-state kinetic assays were performed using magnesium deuteroporphyrin IX (MgD), a more water-soluble substrate analogue of MgP. Initial rate studies showed that the reaction proceeds via a ternary complex. Product (S-adenosyl-L-homocysteine; SAH) inhibition was used to investigate the kinetic mechanism further. SAH was shown to exhibit competitive inhibition with respect to SAM, and mixed inhibition with respect to MgD. This is indicative of a random binding mechanism, whereby SAH may bind productively to either free enzyme or a ChlM-MgD complex. Our results provide an overview of the steady-state kinetics for this enzyme, which are significant given the role of MgP and MgPME in plastid-to-nucleus signalling and their likely critical role in the regulation of this biosynthetic pathway.
镁原卟啉IX甲基转移酶（ChlM）催化镁原卟啉IX（MgP）在C（6）丙酸侧链上的甲基化反应，形成镁原卟啉IX单甲酯（MgPME）。基于序列相似性和预测二级结构的穿线法已被用于将该酶归类到特定类别的S-腺苷-L-甲硫氨酸（SAM）结合蛋白中。这些搜索表明，ChlM含有一个七链β-折叠，这在小分子甲基转移酶中很常见。使用镁中卟啉IX（MgD）进行稳态动力学测定，MgD是一种水溶性更好的MgP底物类似物。初始速率研究表明，该反应通过三元复合物进行。产物（S-腺苷-L-高半胱氨酸；SAH）抑制作用被用于进一步研究动力学机制。结果表明，SAH对SAM表现出竞争性抑制，对MgD表现出混合型抑制。这表明存在一种随机结合机制，即SAH可能有效地结合到游离酶或ChlM-MgD复合物上。我们的结果概述了该酶的稳态动力学，鉴于MgP和MgPME在质体到细胞核信号传导中的作用以及它们在该生物合成途径调控中可能的关键作用，这些结果具有重要意义。
