Feng Z, Aggeler R, Haughton M A, Capaldi R A
Institute of Molecular Biology, University of Oregon, Eugene, 97403-1229, USA.
J Biol Chem. 1996 Jul 26;271(30):17986-9. doi: 10.1074/jbc.271.30.17986.
Cys-87, one of two intrinsic cysteines of the gamma subunit of the Escherichia coli ATP synthase (ECF1F0), is in a short segment of this subunit that binds to the bottom domain of a beta subunit close to a glutamate (Glu-381). Cys-87 was unreactive to maleimides under all conditions in wild-type ECF1 and ECF1F0 but became reactive when Glu-381 of beta was replaced by a cysteine or alanine. The reactivity of Cys-87 with maleimides was nucleotide-dependent, occurring with ATP or ADP + EDTA in catalytic sites, in the presence of AMP.PNP + Mg2+ but not with ADP + Mg2+ bound, whether Pi was present or not, and not when nucleotide binding sites were empty. Binding of N-ethylmaleimide had no effect, whereas 7-diethyl-amino-3-(4'-maleimidylphenyl)-4-methylcoumarin increased the ATPase activity of ECF1 more than 2-fold by reaction with Cys-87. In ECF1F0, these reagents inhibited activity. The nucleotide dependence of the reaction of Cys-87 of the gamma subunit depended on the presence of the epsilon subunit. In epsilon subunit-free ECF1, maleimides reacted with Cys-87 under all nucleotide conditions, including when catalytic sites were empty. These results are discussed in terms of nucleotide-dependent movements of the gamma subunit during functioning of the F1F0-type ATPase.
半胱氨酸-87是大肠杆菌ATP合酶（ECF1F0）γ亚基的两个内在半胱氨酸之一，位于该亚基的一个短片段中，该片段与靠近谷氨酸（Glu-381）的β亚基底部结构域结合。在野生型ECF1和ECF1F0的所有条件下，半胱氨酸-87对马来酰亚胺无反应，但当β亚基的Glu-381被半胱氨酸或丙氨酸取代时，它变得有反应。半胱氨酸-87与马来酰亚胺的反应性是核苷酸依赖性的，在催化位点与ATP或ADP + EDTA一起出现，在存在AMP.PNP + Mg2+的情况下，但不存在结合的ADP + Mg2+时，无论是否存在无机磷酸，并且当核苷酸结合位点为空时不发生反应。N-乙基马来酰亚胺的结合没有影响，而7-二乙氨基-3-（4'-马来酰亚胺基苯基）-4-甲基香豆素通过与半胱氨酸-87反应使ECF1的ATP酶活性增加了2倍以上。在ECF1F0中，这些试剂抑制活性。γ亚基半胱氨酸-87反应的核苷酸依赖性取决于ε亚基的存在。在无ε亚基的ECF1中，马来酰亚胺在所有核苷酸条件下与半胱氨酸-87反应，包括催化位点为空时。根据F1F0型ATP酶功能期间γ亚基的核苷酸依赖性运动对这些结果进行了讨论。
