Phosphorylation of the sarcoplasmic reticulum Ca(2+)-ATPase from ATP and ATP analogs studied by infrared spectroscopy.
Phosphorylation of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) was studied with time-resolved Fourier transform infrared spectroscopy. ATP and ATP analogs (ITP, 2'- and 3'-dATP) were used to study the effect of the adenine ring and the ribose hydroxyl groups on ATPase phosphorylation. All modifications of ATP altered conformational changes and phosphorylation kinetics. The differences compared with ATP increased in the following order: 3'-dATP > ITP > 2'-dATP. Enzyme phosphorylation with ITP results in larger absorbance changes in the amide I region, indicating larger conformational changes of the Ca(2+)-ATPase. The respective absorbance changes obtained with 3'-dATP are significantly different from the others with different band positions and amplitudes in the amide I region, indicating different conformational changes of the protein backbone. ATPase phosphorylation with 3'-dATP is also much ( approximately 30 times) slower than with ATP. Our results indicate that modifications to functional groups of ATP (the ribose 2'- and 3'-OH and the amino group in the adenine ring) affect gamma-phosphate transfer to the phosphorylation site of the Ca(2+)-ATPase by changing the extent of conformational change and the phosphorylation rate. ADP binding to the ADP-sensitive phosphoenzyme (Ca(2)E1P) stabilizes the closed conformation of Ca(2)E1P.
Liu
Man
M
Institut für Biophysik, Johann Wolfgang Goethe-Universität, 60596 Frankfurt am Main, Germany. liu@dbb.su.se
Barth
Andreas
A
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D001081:Apyrase / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D012519:Sarcoplasmic Reticulum / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D017550:Spectroscopy, Fourier Transform Infrared
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001081:Apyrase; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases