pH and magnesium dependence of ATP binding to sarcoplasmic reticulum ATPase. Evidence that the catalytic ATP-binding site consists of two domains.
Nucleotide binding to sarcoplasmic reticulum vesicles was investigated in the absence of calcium using both filtration and fluorescence measurements. Filtration assays of binding of radioactive nucleotides at concentrations up to 0.1 mM gave a stoichiometry of one ATP-binding site/sarcoplasmic reticulum ATPase molecule. When measured in the presence of calcium under otherwise similar conditions, ATPase velocity rose 4-8-fold (depending on pH and magnesium concentration) when the ATP concentration was increased from 1 microM to 0.1 mM. Binding of ATP and ADP enhanced the intrinsic fluorescence of sarcoplasmic reticulum ATPase, but AMP and adenosine did not affect it. Both filtration and fluorescence measurements showed that binding of metal-free ATP is independent of pH (Kd = 20-25 microM) but that the presence of magnesium induces pH dependence of the binding of the Mg.ATP complex (Kd = 10 microM at pH 6.0 and 1.5 microM at pH 8.0). Binding of metal-free ADP was pH-dependent but was not affected by magnesium. High magnesium concentrations inhibited nucleotide binding. These results suggest that ATP interacts with two different domains of Ca-ATPase that form the catalytic site. The first domain may bind the adenine moiety of the substrate, and the pH dependence of ADP binding suggests the participation of His683 in this region. The second domain of the catalytic site may bind the gamma-phosphate and the magnesium ion of the Mg.ATP complex and constitute the locus of the electrostatic interactions between the substrate and the enzyme.
Lacapère
J J
JJ
Département de Biologie, Centre National de la Recherche Scientifique (URA 1290), Gif-sur-Yvette, France.
Bennett
N
N
Dupont
Y
Y
Guillain
F
F
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology*; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D014462:Ultrafiltration
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D004121:Dimethyl Sulfoxide