ADP-binding and ATP-binding sites in native and proteinase-K-digested creatine kinase, probed by reaction-induced difference infrared spectroscopy.
Conformational changes induced by nucleotide binding to native creatine kinase (CK) from rabbit muscle and to proteinase-K-digested (nicked) CK, were investigated by infrared spectroscopy. Photochemical release of ATP from ATP[Et(PhNO2)] in the presence of creatine and native CK produced reaction-induced difference infrared spectra (RIDS) of CK related to structural changes of the enzyme that paralleled the reversible phosphoryl transfer from ATP to creatine. Similarly the photochemical release of ADP from ADP[Et(PhNO2)] in the presence of phosphocreatine and native CK allowed us to follow the backward reaction and its corresponding RIDS. Infrared spectra of native CK indicated that carboxylate groups of Asp or Glu, and some carbonyl groups of the peptide backbone are involved in the enzymatic reaction. Native and proteinase nicked CK have similar Stokes' radii, tryptophan fluorescence, fluorescence fraction accessible to iodide, and far-ultraviolet CD spectra, indicating that native and modified enzymes have the same quaternary structures. However, infrared data showed that the binding site of the gamma-phosphate group of the nucleotide was affected in nicked CK compared with that of the native CK. Furthermore, the infrared absorptions associated with ionized carboxylate groups of Asp or Glu amino acid residues were different in nicked CK and in native CK.
Raimbault
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Laboratoire de Physico-Chimie Biologique, Université Claude Bernard-Lyon I, UFR de Chimie-Biochimie, Villeurbanne, France.
Clottes
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E
Leydier
C
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Vial
C
C
Buchet
R
R
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D003401:Creatine / Q000378:metabolism; D003402:Creatine Kinase / Q000378:metabolism*; D019286:Endopeptidase K / Q000378:metabolism*; D006868:Hydrolysis; D007527:Isoenzymes; D009132:Muscles / Q000201:enzymology; D010710:Phosphates / Q000378:metabolism; D011817:Rabbits; D017550:Spectroscopy, Fourier Transform Infrared
D007527:Isoenzymes; D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D003402:Creatine Kinase; D019286:Endopeptidase K; D003401:Creatine