Investigation of the role of the domain linkers in separate site catalysis by Clostridium symbiosum pyruvate phosphate dikinase.
Investigation of the role of the domain linkers in separate site catalysis by Clostridium symbiosum pyruvate phosphate dikinase.
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研究结构域接头在共生梭菌丙酮酸磷酸二激酶的单独位点催化中的作用。
DOI：
10.1021/bi0113061
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Dunaway-Mariano,D
中科院分区：
文献类型：
--
作者：
Wei,M;Ye,D;Dunaway-Mariano,D
丙酮酸磷酸二激酶 (PPDK) 使用 Mg2+ 和 NH4+ 离子作为辅助因子，催化可逆反应： ATP + Pi+ 丙酮酸 ↔ AMP + PPi+ PEP。该反应分三个步骤进行，每个步骤由位于该三结构域酶的中心结构域表面上的载体组氨酸残基介导： （1）E-His + ATP ↔ E-His-PP·AMP，（2）E-His-PP·AMP + Pi↔ E-His-P + AMP + PPi，（3）E-His-P + 丙酮酸↔ E-His + PEP。前两个部分反应在位于N端结构域的活性位点催化，第三部分反应在位于C端结构域的活性位点催化。对于催化周转，中心域从一个末端域移动到另一末端域。这项工作的目标是确定两个连接接头是否在催化周转过程中指导中心结构域在活性位点之间的移动。该酶的 X 射线晶体结构表明两个连接体之间的相互作用可能导致它们的协调运动。在连接子上进行突变的目的是破坏连接子之间的相互作用，从而破坏同步的连接子运动。分析了五个接头突变体。其中两个在接头的溶剂化区域内包含 4-Ala 插入，三个在该区域具有 3 个残基缺失。测量突变体催化完全反应以及 N 端结构域的 E-His + ATP ↔ E-His-PP·AMP 部分反应和 C 端结构域的 E-His + PEP ↔ E-His-P + 丙酮酸反应的效率，以评估接头功能。三个接头突变体在两个活性位点均具有高活性催化剂，第四个接头突变体在一个位点具有高活性，但在另一个位点则不具有高活性。这些结果被解释为反对协调链接子移动的证据，并表明当中心域在活性位点之间移动时，链接子独立移动。据推测，虽然接头在中心域-末端域对接中发挥被动作用，但它们的结构设计最大限度地减少了扩散过程中搜索的构象空间。
Pyruvate phosphate dikinase (PPDK) catalyzes the reversible reaction: ATP + Pi+ pyruvate ↔ AMP + PPi+ PEP using Mg2+and NH4+ions as cofactors. The reaction takes place in three steps, each mediated by a carrier histidine residue located on the surface of the central domain of this three-domain enzyme: (1) E-His + ATP ↔ E-His-PP·AMP, (2) E-His-PP·AMP + Pi↔ E-His-P + AMP + PPi, (3) E-His-P + pyruvate ↔ E-His + PEP. The first two partial reactions are catalyzed at an active site located on the N-terminal domain, and the third partial reaction is catalyzed at an active site located on the C-terminal domain. For catalytic turnover, the central domain travels from one terminal domain to the other. The goal of this work is to determine whether the two connecting linkers direct the movement of the central domain between active sites during catalytic turnover. The X-ray crystal structure of the enzyme suggests interaction between the two linkers that may result in their coordinated movement. Mutations were made at the linkers for the purpose of disrupting the linker−linker interaction and, hence, synchronized linker movement. Five linker mutants were analyzed. Two of these contain 4-Ala insertions within the solvated region of the linker, and three have 3-residue deletions in this region. The efficiencies of the mutants for catalysis of the complete reaction as well as the E-His + ATP ↔ E-His-PP·AMP partial reaction at the N-terminal domain and the E-His + PEP ↔ E-His-P + pyruvate reaction at the C-terminal domain were measured to assess linker function. Three linker mutants are highly active catalysts at both active sites, and the fourth is highly active at one site but not the other. These results are interpreted as evidence against coordinated linker movement, and suggest instead that the linkers move independently as the central domain travels between active sites. It is hypothesized that while the linkers play a passive role in central domain−terminal domain docking, their structural design minimizes the conformational space searched in the diffusion process.