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Effect of Structure Variations in the Inter-subunit Contact Zone on the Activity and Allosteric Regulation of Inorganic Pyrophosphatase from Mycobacterium tuberculosis
Biochemistry (Moscow) ( IF 2.3 ) Pub Date : 2020-03-01 , DOI: 10.1134/s0006297920030086
R S Romanov 1 , S A Kurilova 1 , A A Baykov 1 , E V Rodina 2
Affiliation  

Hexameric inorganic pyrophosphatase from Mycobacterium tuberculosis (Mt-PPase) has a number of structural and functional features that distinguish it from homologous enzymes widely occurring in living organisms. In particular, it has unusual zones of inter-subunit contacts and lacks the N-terminal region common for other PPases. In this work, we constructed two mutant forms of the enzyme, Ec-Mt-PPase and R14Q-Mt-PPase. In Ec-Mt-PPase, the missing part of the polypeptide chain was compensated with a fragment of PPase from Escherichia coli (Ec-PPase). In R14Q-Mt-PPase, a point mutation was introduced to the contact interface between the two trimers of the hexamer. Both modifications significantly improved the catalytic activity of the enzyme and abolished its inhibition by the cofactor (Mg2+ ion) excess. Activation of Mt-PPase by low (∼10 µM) concentrations of ATP, fructose-1-phosphate, L-malate, and non-hydrolyzable substrate analogue methylene bisphosphonate (PCP) was observed. At concentrations of 100 µM and higher, the first three compounds acted as inhibitors. The activating effect of PCP was absent in both mutant forms, and the inhibitory effect of fructose-1-phosphate was absent in Ec-Mt-PPase. The effects of other modulators varied only quantitatively among the mutants. The obtained data indicate the presence of allosteric sites in Mt-PPase, which are located in the zones of inter-subunit contact or associated with them.

中文翻译:

亚基间接触区结构变异对结核分枝杆菌无机焦磷酸酶活性和变构调控的影响

来自结核分枝杆菌的六聚无机焦磷酸酶 (Mt-PPase) 具有许多结构和功能特征,使其有别于广泛存在于活生物体中的同源酶。特别是,它具有不寻常的亚基间接触区,并且缺乏其他 PPase 常见的 N 端区域。在这项工作中,我们构建了两种突变形式的酶,Ec-Mt-PPase 和 R14Q-Mt-PPase。在 Ec-Mt-PPase 中,多肽链的缺失部分由来自大肠杆菌的 PPase 片段 (Ec-PPase) 补偿。在 R14Q-Mt-PPase 中,在六聚体的两个三聚体之间的接触界面上引入了点突变。两种修饰都显着提高了酶的催化活性,并消除了辅因子(Mg2+ 离子)过量的抑制作用。观察到低 (~10 µM) 浓度的 ATP、1-磷酸果糖、L-苹果酸和不可水解底物类似物亚甲基双膦酸盐 (PCP) 激活 Mt-PPase。在 100 µM 和更高的浓度下,前三种化合物充当抑制剂。两种突变体均不存在 PCP 的激活作用,而 Ec-Mt-PPase 中不存在 1-磷酸果糖的抑制作用。其他调节剂的作用在突变体之间仅在数量上有所不同。获得的数据表明 Mt-PPase 中存在变构位点,这些位点位于亚基间接触区域或与之相关。两种突变体均不存在 PCP 的激活作用,而 Ec-Mt-PPase 中不存在 1-磷酸果糖的抑制作用。其他调节剂的作用在突变体之间仅在数量上有所不同。获得的数据表明 Mt-PPase 中存在变构位点,这些位点位于亚基间接触区域或与之相关。两种突变体均不存在 PCP 的激活作用,而 Ec-Mt-PPase 中不存在 1-磷酸果糖的抑制作用。其他调节剂的作用在突变体之间仅在数量上有所不同。获得的数据表明 Mt-PPase 中存在变构位点,这些位点位于亚基间接触区域或与之相关。
更新日期:2020-03-01
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