Digel J G, McCarty R E
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Biochemistry. 1995 Nov 7;34(44):14482-9. doi: 10.1021/bi00044a026.
Chloroplast coupling factor 1 (CF1) deficient in its epsilon subunit was loaded with 2'(3')-O-trinitrophenyl-ADP (TNP-ADP), and the release of tightly bound TNP-ADP was followed as a decrease in fluorescence. TNP-ADP could be exchanged for medium ADP, ATP, MgADP, and MgATP. The preferred substrate for exchange was MgADP, particularly in the presence of P(i). One nucleotide binding site contained ADP which was not displaced during TNP-ADP loading. When Mg2+ was bound at this site, complete exchange of bound TNP-ADP for medium nucleotide was prevented. This tightly bound MgADP was removed by incubation of the enzyme with EDTA. Tightly bound TNP-ADP was removed by high concentrations of sulfite, sulfate, or P(i) in the absence of medium nucleotide and free Mg2+, regardless of the bound Mg2+ content of the enzyme. Sulfite and P(i), in the presence of medium nucleotide and Mg2+, enabled complete exchange of tightly bound TNP-ADP. The combination of Mg2+ and sulfite, or Mg2+ and P(i), caused exchange of tightly bound ADP from two different sites. These results suggest that both sites exchange when the enzyme is fully active, and that at least three sites are likely to participate in catalysis.
缺失ε亚基的叶绿体偶联因子1（CF1）被加载了2'（3'）-O-三硝基苯基-ADP（TNP-ADP），随着荧光的降低监测紧密结合的TNP-ADP的释放。TNP-ADP可以与介质中的ADP、ATP、MgADP和MgATP进行交换。交换的首选底物是MgADP，特别是在有磷酸根（P(i)）存在的情况下。一个核苷酸结合位点含有ADP，在加载TNP-ADP期间不会被取代。当Mg2+结合在该位点时，结合的TNP-ADP与介质核苷酸的完全交换被阻止。通过将酶与EDTA孵育可去除这种紧密结合的MgADP。在没有介质核苷酸和游离Mg2+的情况下，高浓度的亚硫酸盐、硫酸盐或磷酸根（P(i)）可去除紧密结合的TNP-ADP，而不管酶结合的Mg2+含量如何。在介质核苷酸和Mg2+存在的情况下，亚硫酸盐和磷酸根（P(i)）可使紧密结合的TNP-ADP完全交换。Mg2+与亚硫酸盐或Mg2+与磷酸根（P(i)）的组合会导致紧密结合的ADP从两个不同位点进行交换。这些结果表明，当酶完全活跃时，两个位点都会进行交换，并且至少有三个位点可能参与催化作用。
