CHANGES OF PROTEIN-STRUCTURE, NUCLEOTIDE MICROENVIRONMENT, AND CA2-BINDING STATES IN THE CATALYTIC CYCLE OF SARCOPLASMIC-RETICULUM CA2+-ATPASE - INVESTIGATION OF NUCLEOTIDE-BINDING, PHOSPHORYLATION AND PHOSPHOENZYME CONVERSION BY FTIR DIFFERENCE SPECTROSCOPY()

Changes of infrared absorbance of sarcoplasmic reticulum Ca2+-ATPase ( EC 3.6.1.38) associated with partial reactions of its catalytic cycle were investigated in the region from 1800 to 950 cm(-1) in H2O and (H2 O)-H-2. Starting from Ca(2)E(1), 3 reaction steps were induced in the infrared cuvette via photolytic release of ATP and ADP: (a) nucleotide binding, (b) formation of the ADP-sensitive phosphoenzyme (Ca(2)E(1)P ) and (c) formation of the ADP-insensitive phosphoenzyme (E(2)P). All reaction steps caused distinct changes of the infrared spectrum which were characteristic for each reaction step but comparable for all step s in the number and magnitude of the changes. Most pronounced were abs orbance changes in the amide I spectral region sensitive to protein se condary structure. However, they were small - less than 1% of the tota l protein absorbance - indicating that the reaction steps are associat ed with small and local conformational changes of the polypeptide back bone instead of a large conformational rearrangement. Especially, ther e is no outstanding conformational change associated with the phosphoe nzyme conversion Ca(2)E(1)P --> E(2)P. ADP-binding induces conformatio nal changes in the ATPase polypeptide backbone with a-helical structur es and presumably beta-sheet or beta-turn structures involved. Phospho rylation is accompanied by the appearance of a keto group vibration th at can tentatively be assigned to the phosphorylated residue Asp(351). Phosphoenzyme conversion and Ca2+-release produce difference signals which can be explained by the release of Ca2+ from carboxylate groups and a change of hydrogen bonding or protonation state of carboxyl grou ps.