Sewell Edward W C, Pereira Mark P, Brown Eric D
Department of Biochemistry and Biomedical Sciences and the Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario L8N 3Z5, Canada.
J Biol Chem. 2009 Aug 7;284(32):21132-8. doi: 10.1074/jbc.M109.010215. Epub 2009 Jun 11.
Wall teichoic acids are a chemically diverse group of anionic polymers that constitute up to 50% of the Gram-positive cell wall. These polymers play a pivotal role in virulence and have been implicated in a diverse range of physiological functions. The TagF-like family of enzymes has been shown to be responsible for wall teichoic acid priming and polymerization events. Although many such enzymes are well validated therapeutic targets, a mechanistic understanding of this enzyme family has remained elusive. TagF is the prototypical teichoic acid polymerase and uses CDP-glycerol to catalyze synthesis of the linear (1,3)-linked poly(glycerol phosphate) teichoic acid in Bacillus subtilis 168. Here we used a synthetic soluble analog of the natural substrate of the enzyme, Lipid , to conduct the first detailed mechanistic investigation of teichoic acid polymerization. Through the use of a new high pressure liquid chromatography-based assay to monitor single glycerol phosphate incorporations into the Lipid analog, we conducted a detailed analysis of reaction product formation patterns and unequivocally showed TagF to be non-processive in vitro. Furthermore by monitoring the kinetics of polymerization, we showed that Lipid analog species varying in size have the same K(m) value of 2.6 microm and validated use of Bi Bi velocity expressions to model the TagF enzyme system. Initial rate analysis showed that TagF catalyzes a sequential Bi Bi mechanism where both substrates are added to the enzyme prior to product release consistent with a single displacement chemical mechanism.
壁磷壁酸是一类化学性质多样的阴离子聚合物，占革兰氏阳性菌细胞壁成分的50%。这些聚合物在致病性方面起着关键作用，并涉及多种生理功能。已证明TagF样酶家族负责壁磷壁酸的起始和聚合过程。尽管许多此类酶是经过充分验证的治疗靶点，但对该酶家族的作用机制仍不清楚。TagF是典型的磷壁酸聚合酶，在枯草芽孢杆菌168中利用CDP -甘油催化合成线性（1,3）-连接的聚（甘油磷酸）磷壁酸。在此，我们使用该酶天然底物的合成可溶性类似物脂质，对磷壁酸聚合进行了首次详细的作用机制研究。通过使用基于高压液相色谱的新测定法来监测单个甘油磷酸掺入脂质类似物的情况，我们对反应产物形成模式进行了详细分析，并明确表明TagF在体外是非连续的。此外，通过监测聚合动力学，我们表明不同大小的脂质类似物物种具有相同的2.6微摩尔的K(m)值，并验证了使用双底物双产物速度表达式来模拟TagF酶系统。初始速率分析表明，TagF催化一种顺序双底物双产物机制，即两种底物在产物释放之前都添加到酶上，这与单取代化学机制一致。
