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J. Biol. Chem., Vol. 279, Issue 48, 49902-49909, November 26, 2004

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Phosphorylation of the Sarcoplasmic Reticulum Ca²+-ATPase from ATP and ATP Analogs Studied by Infrared Spectroscopy^*

Man Liu and Andreas Barth¶

From the Institut für Biophysik, Johann Wolfgang Goethe-Universität, 60596 Frankfurt am Main, Germany and ^¶Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden

Phosphorylation of the sarcoplasmic reticulum Ca²⁺-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 Ca²⁺-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 (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 -phosphate transfer to the phosphorylation site of the Ca²⁺-ATPase by changing the extent of conformational change and the phosphorylation rate. ADP binding to the ADP-sensitive phosphoenzyme (Ca₂E1P) stabilizes the closed conformation of Ca₂E1P.

Received for publication, July 16, 2004 , and in revised form, September 7, 2004.

^* This work was supported by Deutsche Forschungsgemeinschaft Grants Ba1887/2-1 and Ma1054/18-3, Vetenskapsrådet, and Knut och Alice Wallenbergs Stiftelse. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 46-8-163529; Fax: 46-8-155597; E-mail: liu{at}dbb.su.se.

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This article has been cited by other articles:

				M. Liu, M. Krasteva, and A. Barth Interactions of Phosphate Groups of ATP and Aspartyl Phosphate with the Sarcoplasmic Reticulum Ca2+-ATPase: An FTIR Study Biophys. J., December 1, 2005; 89(6): 4352 - 4363. [Abstract] [Full Text] [PDF]