Subunit-subunit interactions in TF1 as revealed by ligand binding to isolated and integrated α and β subunits

Matthias Rögner, Peter Gräber, Uwe Lücken, Henri Tiedge, Joachim Weber, Günter Schäfer

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

The binding of 3′-O-(1-naphthoyl)adenosinetriphosphate (1-naphthoyl-ATP), ATP and ADP to TF1 and to the isolated α and β subunits was investigated by measuring changes of intrinsic protein fluorescence and of fluorescence anisotropy of 1-naphthoyl-ATP upon binding. The following results were obtained. (1) The isolated α and β subunits bind 1 mol 1-naphthoyl-ATP with a dissociation constant (KD(1-naphthoyl-ATP)) of 4.6 μM and 1.9 μM, respectively. (2) The KD(ATP) for α and β subunits is 8 μM and 11 μM, respectively. (3) The KD(ADP) for α and β subunits is 38 μM μM and 7 μM, respectively. (4) TF1 binds 2 mol 1-naphthoyl-ATP per mol enzyme with KD = 170 nM. (5) The rate constant for 1-naphthoyl-ATP binding to α and β subunit is more than 5 · 104 M-1s-1. (6) The rate constant for 1-naphthoyl-ATP binding to TF1 is 6.6 · 103 M-1 · s-1 (monophasic reaction); the rate constant for its dissociation in the presence of ATP is biphasic with a fast first phase (kA -1 = 3 · 10-3 s-1) and a slower second phase (kA -2 < 0.2 · 10-3 s-1). From the appearance of a second peak in the fluorescence emission spectrum of 1-naphthoyl-ATP upon binding it is concluded that the binding sites in TF1 are located in an environment more hydrophobic than the binding sites on isolated α and β subunits. The differences in kinetic and thermodynamic parameters for ligand binding to isolated versus integrated α and β subunits, respectively, are explained by interactions between these subunits in the enzyme complex.

Original languageEnglish
Pages (from-to)121-130
Number of pages10
JournalBBA - Bioenergetics
Volume849
Issue number1
DOIs
StatePublished - Apr 2 1986

Keywords

  • (Thermophilic bacterium PS3)
  • ATPase
  • Fluorescence
  • Nucleotide analogue
  • Nucleotide binding
  • Subunit interaction

Fingerprint Dive into the research topics of 'Subunit-subunit interactions in TF<sub>1</sub> as revealed by ligand binding to isolated and integrated α and β subunits'. Together they form a unique fingerprint.

  • Cite this