Role of βAsn-243 in the phosphate-binding subdomain of catalytic sites of Escherichia coli F1-ATPase
Role of βAsn-243 in the phosphate-binding subdomain of catalytic sites of Escherichia coli F1-ATPase
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DOI：
10.1074/jbc.m407608200
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发表时间：
2004-10-29
影响因子：
4.8
通讯作者：
Senior, AE
中科院分区：
文献类型：
--
作者：
Ahmad, Z;Senior, AE
在ATP合成酶的催化机制中，磷酸(P-I)的结合和释放步骤被认为与伽马亚基旋转相关，P-I结合被认为是面对高细胞[ATP]/[ADP]比率结合ADP的先决条件。在X射线结构中，βAsn-243残基位于催化中心的P-I结合亚区。在这里，我们通过对Ala和Asp的突变，研究了BetaAsn-243在大肠杆菌ATP合成酶中的作用。突变BetaN243A导致F-1-ATPase活性降低30倍；7-氯-4-硝基苯并-2-氧-1，3-二唑对该活性的抑制作用弱于野生型，并且P-I受到保护。ADP-氟铝酸盐的抑制作用强于野生型，而ADP-氟化镓的抑制作用弱于野生型。βN243D F-1-ATPase活性降低1300倍，且不受ADP-氟铝酸盐或ADP-氟化镓的抑制。7-氯-4-硝基苯并-2-氧杂-1，3-二唑激活了β-N243D F-1-ATPase，P-I不影响激活。我们得出结论，BetaAsn-243残基不直接参与P-I结合，但通过广泛参与与邻近残基的氢键，是正确组织过渡态复合体所必需的。它还可能涉及“攻击水”和与之相关的第二水的定向。
In the catalytic mechanism of ATP synthase, phosphate (P-i) binding and release steps are believed to be correlated to gamma-subunit rotation, and P-i binding is proposed to be prerequisite for binding ADP in the face of high cellular [ATP]/[ADP] ratios. In x-ray structures, residue betaAsn-243 appears centrally located in the P-i-binding subdomain of catalytic sites. Here we studied the role of betaAsn-243 in Escherichia coli ATP synthase by mutagenesis to Ala and Asp. Mutation betaN243A caused 30-fold impairment of F-1-ATPase activity; 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole inhibited this activity less potently than in wild type and P-i protected from inhibition. ADP-fluoroaluminate was more inhibitory than in wild-type, but ADP-fluoroscandium was less inhibitory. betaN243D F-1-ATPase activity was impaired by 1300-fold and was not inhibited by ADP-fluoroaluminate or ADP-fluoroscandium. 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole activated betaN243D F-1-ATPase, and P-i did not affect activation. We conclude that residue betaAsn-243 is not involved in P-i binding directly but is necessary for correct organization of the transition state complex through extensive involvement in hydrogen bonding to neighboring residues. It is also probably involved in orientation of the "attacking water" and of an associated second water.