Changes of protein structure, nucleotide microenvironment, and Ca(2+)-binding states in the catalytic cycle of sarcoplasmic reticulum Ca(2+)-ATPase: investigation of nucleotide binding, phosphorylation and phosphoenzyme conversion by FTIR difference spectroscopy.
Changes of infrared absorbance of sarcoplasmic reticulum Ca(2+)-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 2H2O. Starting from Ca2E1, 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 (Ca2E1P) and (c) formation of the ADP-insensitive phosphoenzyme (E2P). All reaction steps caused distinct changes of the infrared spectrum which were characteristic for each reaction step but comparable for all steps in the number and magnitude of the changes. Most pronounced were absorbance changes in the amide I spectral region sensitive to protein secondary structure. However, they were small--less than 1% of the total protein absorbance--indicating that the reaction steps are associated with small and local conformational changes of the polypeptide backbone instead of a large conformational rearrangement. Especially, there is no outstanding conformational change associated with the phosphoenzyme conversion Ca2E1P-->E2P. ADP-binding induces conformational changes in the ATPase polypeptide backbone with alpha-helical structures and presumably beta-sheet or beta-turn structures involved. Phosphorylation is accompanied by the appearance of a keto group vibration that can tentatively be assigned to the phosphorylated residue Asp351. Phosphoenzyme conversion and Ca(2+)-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 groups.
Barth
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Institut für Biophysik, Strahlenbiologie der Universität Freiburg, Freiburg, Germany.
Kreutz
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Mäntele
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D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D002118:Calcium / Q000737:chemistry; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D017666:Deuterium Oxide; D008956:Models, Chemical; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013055:Spectrophotometry, Infrared / Q000379:methods; D014867:Water
D009711:Nucleotides; D011506:Proteins; D014867:Water; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D017666:Deuterium Oxide; D002118:Calcium