Structural changes of the sarcoplasmic reticulum Ca(2+)-ATPase upon nucleotide binding studied by fourier transform infrared spectroscopy.
Changes in the vibrational spectrum of the sarcoplasmic reticulum Ca(2+)-ATPase upon nucleotide binding were recorded in H(2)O and (2)H(2)O at -7 degrees C and pH 7.0. The reaction cycle was triggered by the photochemical release of nucleotides (ATP, ADP, and AMP-PNP) from a biologically inactive precursor (caged ATP, P(3)-1-(2-nitrophenyl) adenosine 5'-triphosphate, and related caged compounds). Infrared absorbance changes due to ATP release and two steps of the Ca(2+)-ATPase reaction cycle, ATP binding and phosphorylation, were followed in real time. Under the conditions used in our experiments, the rate of ATP binding was limited by the rate of ATP release (k(app) congruent with 3 s(-1) in H(2)O and k(app) congruent with 7 s(-1) in (2)H(2)O). Bands in the amide I and II regions of the infrared spectrum show that the conformation of the Ca(2+)-ATPase changes upon nucleotide binding. The observation of bands in the amide I region can be assigned to perturbations of alpha-helical and beta-sheet structures. According to similar band profiles in the nucleotide binding spectra, ATP, AMP-PNP, and ADP induce similar conformational changes. However, subtle differences between ATP and AMP-PNP are observed; these are most likely due to the protonation state of the gamma-phosphate group. Differences between the ATP and ADP binding spectra indicate the significance of the gamma-phosphate group in the interactions between the Ca(2+)-ATPase and the nucleotide. Nucleotide binding affects Asp or Glu residues, and bands characteristic of their protonated side chains are observed at 1716 cm(-1) (H(2)O) and 1706 cm(-1) ((2)H(2)O) and seem to depend on the charge of the phosphate groups. Bands at 1516 cm(-1) (H(2)O) and 1514 cm(-1) ((2)H(2)O) are tentatively assigned to a protonated Tyr residue affected by nucleotide binding. Possible changes in Arg, Trp, and Lys absorption and in the nucleoside are discussed. The spectra are compared with those of nucleotide binding to arginine kinase, creatine kinase, and H-ras P21.
von Germar
F
F
Institut für Biophysik, Johann Wolfgang Goethe Universität Frankfurt, D-60590 Frankfurt am Main, Germany.
Barth
A
A
Mäntele
W
W
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000493:pharmacokinetics; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000493:pharmacokinetics; D000266:Adenylyl Imidodiphosphate / Q000493:pharmacokinetics; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D017666:Deuterium Oxide; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009578:Nitrobenzenes / Q000493:pharmacokinetics; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D014867:Water
D009578:Nitrobenzenes; C101520:P(2)-(1-(2-nitrophenyl)ethyl) ADP; D014867:Water; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; C029273:P(3)-1-(2-nitro)phenylethyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D017666:Deuterium Oxide