Allosteric effect of ATP on Na(+),K(+)-ATPase conformational kinetics.
The kinetics of the E2 --> E1 conformational change of unphosphorylated Na+,K+-ATPase was investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24 degrees C). The enzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize the E2 conformation. When rabbit enzyme was mixed with 130 mM NaCl alone or with 130 mM NaCl and varying concentrations of Na2ATP simultaneously, a fluorescence decrease was observed. In the absence of ATP, the fluorescence decrease followed a biexponential time course, but at ATP concentrations after mixing of >or=50 microM, the fluorescence transient could be adequately fitted by a single exponential. On the basis of the agreement between theoretical simulations and experimental traces, we propose that in the absence of bound ATP the conformational transition occurs as a two step reversible process within a protein dimer, E2:E2 --> E2:E1 --> E1:E1. In the presence of 130 mM NaCl, the sum of the forward and backward rate constants for the E2:E2 --> E2:E1 and E2:E1 --> E1:E1 transitions were found to be 10.4 (+/-1.0) and 0.49 (+/-0.02) s-1, respectively. At saturating concentrations of ATP, however, the transition occurs in a single reversible step with the sum of its forward and backward rate constants equal to 35.2 (+/-0.3) s-1. It was found that ATP acting at a high affinity site (Kd approximately 0.25 microM), stimulated the reverse reaction, E1ATP --> E2ATP, in addition to its known allosteric low affinity (Kd approximately 71 microM) stimulation of the forward reaction, E2ATP --> E1ATP.
Clarke
Ronald J
RJ
School of Chemistry, University of Sydney, Sydney, NSW 2006, Australia, and Faculty of Biology, University of Konstanz, D-78435 Konstanz, Germany. r.clarke@chem.usyd.edu.au
Apell
Hans-Jürgen
HJ
Kong
Benjamin Y
BY
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000494:Allosteric Regulation; D000818:Animals; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D010766:Phosphorylation; D011487:Protein Conformation; D011817:Rabbits; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase