ATP-INDUCED PHOSPHORYLATION OF THE SARCOPLASMIC-RETICULUM CA2- MOLECULAR INTERPRETATION OF INFRARED DIFFERENCE SPECTRA( ATPASE )

Time-resolved infrared difference spectra of the ATP-induced phosphory lation of the sarcoplasmic reticulum Ca2+-ATPase have been recorded in H2O and (H2O)-H-2 at pH 7.0 and 1 degrees C. The reaction was induced by ATP release from P-3-1-(2-nitro)phenylethyladenosine 5'-triphospha te (caged ATP) and from [gamma-O-18(3)]caged ATP. A band at 1546 cm(-1 ), not observed with the deuterated enzyme, can be assigned to the ami de II mode of the protein backbone and indicates that a conformational change associated with ATPase phosphorylation takes place after ATP b inding. This is also indicated between 1700 and 1610 cm(-1), where ban dshifts of up to 10 cm(-1) observed upon protein deuteration suggest t hat amide I modes of the protein backbone dominate the difference spec trum. From the band positions it is deduced that alpha-helical, beta-s heet, and probably beta-turn structures are affected in the phosphoryl ation reaction. Model spectra of acetyl phosphate, acetate, ATP, and A DP suggest the tentative assignment of some of the bands of the phosph orylation spectrum to the molecular groups of ATP and Asp(351), which participate directly in the phosphate transfer reaction: a positive ba nd at 1719 cm(-1) to the C=O group of aspartyl phosphate, a negative b and at 1239 cm(-1) to the nu(as)(PO2-) modes of the bound ATP molecule , and a positive band at 1131 cm(-1) to the nu(as)(PO32-) mode of the phosphoenzyme phosphate group, the latter assignment being supported b y the band's sensitivity toward isotopic substitution in the gamma-pho sphate of ATP. Band positions and shapes of these bands indicate that the alpha- and/or beta-phosphate(s) of the bound ATP molecule become p artly dehydrated when ATP binds to the ATPase, that the phosphoenzyme phosphate group is unprotonated at pH 7.0, and that the C=O group of a spartyl phosphate does not interact with bulk water. The Ca2+ binding sites seem to be largely undisturbed by the phosphorylation reaction, and a functional role of the side chains of Asn, Gin, and Arg residues was not detected.