Optimal activity of adenosinetriphosphatase (ATPase) in coupling factor 1 was obtained as a function of divalent metal ion-ATP complex rather than of either free metal or free ATP, indicating that the complex is the true substrate. High concentrations of either the complex, the free ATP, or the free metal ion were competitive inhibitors of ATPase activity. This suggests that the complex is attached through at least two points of attachment to the active site, one through the metal ion and another possibly through the base of the nucleotide. Divalent metal ions that were the best activators were found to be the strongest inhibitors in their free forms. This is seen when the order of with the various complexes (MnATP2- > MgATP2” > CaATP2-) is compared to the order of K for competitive inhibition by the free ions (Ca2+ > Mg2+ > Mn2+). Direct binding studies indicated that CF, has one tight and five loose sites for Mn2+ binding. Various di- or triphosphonucleotides and pyrophosphate induce the appearance of a second tight site and the disappearance of one of the loose sites. It is suggested that the two tight sites are at the active site of the enzyme for the following reasons. (1) The Ki (5µM) for free Mn2+ as a competitive inhibitor of ATPase activity is similar to the Kd (~1 µM) of Mn2+ binding. (2) All the effectors which tighten the binding of Mn2+ are either substrates or competitive inhibitors of ATPase activity. (3) A change from pH 8 to pH 6.5 similarly increases both the K. for binding Mn2+ and its as a competitive inhibitor.
|Number of pages||6|
|State||Published - Oct 1981|