Shapiro A B, McCarty R E
Division of Biological Sciences, Cornell University, Ithaca, New York 14853.
J Biol Chem. 1988 Oct 5;263(28):14160-5.
Fluorescence resonance energy transfer was used to show that ATP hydrolysis induces a change in the properties of two nucleotide-binding sites in isolated chloroplast coupling factor 1 (CF1). The fluorescence donor was Lucifer Yellow vinyl sulfone (4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide- 3,6-disulfonate), covalently bound to a unique site on the alpha subunit between nucleotide-binding sites 2 and 3. The fluorescence acceptor was the ATP analog 2'(3')-trinitrophenyladenosine 5'-triphosphate (TNP-ATP), incorporated specifically into nucleotide-binding site 1. Energy transfer from Lucifer Yellow to TNP-ATP in site 1 was greater if catalysis occurred before TNP-ATP was incorporated than if no catalysis occurred, indicating that one of the nucleotide-binding sites near the Lucifer Yellow had changed its properties to those of site 1 as a result of catalysis. The amount of energy transfer increased with the degree of substrate excess during catalysis, as expected if catalysis were required for the new site 1 location. ADP, which binds to CF1, but is not a substrate for hydrolysis, caused little energy transfer. Titration of site 3 with TNP-ATP showed greater energy transfer from Lucifer Yellow when catalysis had not occurred, indicating that sites 1 and 3 switched properties as a result of catalysis. The amount of energy transfer declined exponentially with time between removal of substrate and addition of TNP-ATP to site 1, with a half-time of 1.5-2 h at room temperature. This result suggests that the change that results in switching of nucleotide-binding sites 1 and 3 relaxes in the absence of substrate. Our results show that the asymmetry of the nucleotide-binding sites of CF1 is not a permanent feature of the molecule.
荧光共振能量转移被用于表明ATP水解会诱导分离的叶绿体偶联因子1（CF1）中两个核苷酸结合位点的性质发生变化。荧光供体是共价结合到α亚基上核苷酸结合位点2和3之间一个独特位点的乙烯砜型荧光素（4-氨基-N-[3-(乙烯磺酰基)phenyl]萘二甲酰亚胺-3,6-二磺酸盐）。荧光受体是特异性掺入核苷酸结合位点1的ATP类似物2'(3')-三硝基苯基腺苷5'-三磷酸（TNP-ATP）。如果在TNP-ATP掺入之前发生催化作用，那么从荧光素到位点1中TNP-ATP的能量转移比未发生催化作用时更大，这表明靠近荧光素的一个核苷酸结合位点由于催化作用而使其性质转变为位点1的性质。能量转移量随着催化过程中底物过量程度的增加而增加，这与新位点1的定位需要催化作用的预期相符。与CF1结合但不是水解底物的ADP几乎不引起能量转移。用TNP-ATP滴定位点3表明，在未发生催化作用时，荧光素的能量转移更大，这表明位点1和3由于催化作用而交换了性质。从去除底物到向位点1添加TNP-ATP之间，能量转移量随时间呈指数下降，在室温下半衰期为1.5 - 2小时。这一结果表明，导致核苷酸结合位点1和3交换的变化在没有底物的情况下会松弛。我们的结果表明，CF1核苷酸结合位点的不对称性不是该分子的一个永久特征。
