Catalytic site nucleotide and inorganic phosphate dependence of the conformation of the epsilon subunit in Escherichia coli adenosinetriphosphatase.
Catalytic site nucleotide and inorganic phosphate dependence of the conformation of the epsilon subunit in Escherichia coli adenosinetriphosphatase.
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大肠杆菌腺苷三磷酸酶中ε亚基构象的催化位点核苷酸和无机磷酸盐依赖性。
DOI：
10.1021/bi00219a017
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Capaldi,RA
中科院分区：
文献类型：
--
作者：
Mendel-Hartvig,J;Capaldi,RA
Janet Mendel-Hartvig和RoderickA. Capaldi* Institute of Molecular Biology，University of俄勒冈州，尤金，俄勒冈州97403接收于1990年7月6日;已经发现大肠杆菌F1的e亚基（ECF 1）的胰蛋白酶切割速率是配体依赖性的，如通过蛋白酶消化时发生的酶的活化间接测量的，或者当直接接着用单克隆抗体监测该亚基的切割时。在单独的ADP、ADP+ Mg ~（2+）、ATP+ EDTA或AMP-PNP存在下，e亚基的切割是快速的，但当Pj与ADP+ Mg ~（2+）一起沿着加入时，或当加入ATP+ Mg ~（2+）以在催化位点产生ADP+ Pj（+Mg ~（2+））时，e亚基的切割是缓慢的。在ADP+ Mg 2+存在下保护e亚基不被胰蛋白酶切割所需的Pj的半最大浓度为50 μ M，这在Pj与Fi的高亲和力结合所测量的范围内。t亚基中的配体依赖性构象变化也在使用水溶性碳二亚胺1-乙基-3-[3-（二甲氨基）丙基]碳二亚胺（EDC）的交联实验中进行了检查。在ATP+ Mg ~（2+）或ADP+ Mg ~（2+）+P_1存在下，e亚基可高产地交联成β_3。在ATP+ EDTA或ADP+ Mg 2+（无Pj）的情况下，β-e交联产物的产率大大降低。我们得出结论，e亚基经历了依赖于Pj的存在下的构象变化。先前已经发现，e亚基与ECFj的结合通过降低Pj的解离速率来降低ATP酶活性[Dunn，S. D、Zadorozny，VD，Tozer，RG，& Orr，L. E.（1987）Biochemistry 26，4488-4493]。结合和E-亚基构象之间的这种相互关系对E. coliATP合成酶。
Janet Mendel-Hartvig and RoderickA. Capaldi* Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403 Received July 6, 1990; Revised Manuscript Received October 17, 1990 abstract; The rate of trypsin cleavage of the e subunit of Escherichia coli F,(ECFj) has been found to be ligand-dependent, as measured indirectly by the activation of the enzyme that occurs on protease digestion, or when followed directly by monitoring the cleavage of this subunit using monoclonal antibodies. The cleavage of the e subunit was fast in the presence of ADP alone, ADP+ Mg2+, ATP+ EDTA, or AMP-PNP, but slow when Pj was added along with ADP+ Mg2+ or when ATP+ Mg2+ was added to generate ADP+ Pj (+ Mg2+) in the catalytic site (s). The half-maximal concentration of Pj required in the presence of ADP+ Mg2+ to protect the e subunit from cleavage by trypsin was 50/uM, which is in the range measured for the high-affinity binding of Pj to F,. The ligand-dependent conformational changes in the t subunit were also examined in cross-linking experiments using the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide (EDC). In the presence of ATP+ Mg2+ or ADP+ Mg2++ Pj, the e subunit cross-linkedto (3 in high yield. With ATP+ EDTA or ADP+ Mg2+(no Pj), the yield of the/3-e cross-linked product was much reduced. We conclude that the e subunit undergoes a conformational change dependent on the presence of Pj. It has been found previously that binding of the e subunit to ECFj inhibitsATPase activity by decreasing the off rate of Pj [Dunn, S. D., Zadorozny, VD, Tozer, RG, & Orr, L. E.(1987) Biochemistry 26, 4488-4493]. This reciprocal relationship between P, binding and e-subunit conformation has important implications for energy transduction by the E. coli ATP synthase.