Structural changes of mitochondrial creatine kinase upon binding of ADP, ATP, or Pi, observed by reaction-induced infrared difference spectra.
Structural modifications of rabbit heart mitochondrial creatine kinase induced by the binding of its nucleotide substrates and Pi were investigated. Reaction-induced difference spectra (RIDS), resulting from the difference between infrared spectra recorded before and after the photorelease of a caged ligand, allow us to detect very small variations in protein structure. Our results indicated that the protein secondary structure remained relatively stable during nucleotide binding. Indeed, this binding to creatine kinase affected only a few amino acids, and caused small peptide backbone deformations and alterations of the carbonyl side chains of aspartate or glutamate, reflecting modifications within preexisting elements rather than a net change in secondary structure. Nonetheless, MgADP and MgATP RIDS were distinct, whereas the MgPi RIDS presented some similarities with the MgATP one. The difference between MgADP and MgATP RIDS could reflect a distinct configuration of the two metal-nucleotide complexes inducing a different positioning and/or a distinct binding mode to the creatine kinase active site. Comparison of the MgATP and MgPi RIDS suggests that Pi binding took place at the same binding site as the gamma-phosphoryl group of ATP. Thus, the difference between MgADP and MgATP RIDS would mainly be due to the effect of the gamma-P of ATP. The differences observed when comparing the RIDS resulting from the binding of nucleotides to octameric mitochondrial creatine kinase or dimeric cytosolic isoform could reflect the distinct oligomerization states and physicochemical or kinetic properties of the two isoenzymes.
Laboratoire de Biomembranes et Enzymes Associés and Laboratoire de Physico-Chimie Biologique, UMR 5013 "Reconnaissance et Transduction Moléculaires", Université Claude Bernard Lyon 1, 69622 Villeurbanne Cedex, France.
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D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D003401:Creatine / Q000378:metabolism; D003402:Creatine Kinase / Q000737:chemistry* / Q000378:metabolism*; D052281:Creatine Kinase, Mitochondrial Form; D004229:Dithiothreitol / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008929:Mitochondria, Heart / Q000201:enzymology*; D009578:Nitrobenzenes / Q000378:metabolism; D028044:Phosphate-Binding Proteins; D010725:Phosphocreatine / Q000378:metabolism; D010782:Photolysis; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D017550:Spectroscopy, Fourier Transform Infrared; D013379:Substrate Specificity
D002352:Carrier Proteins; D007527:Isoenzymes; D009578:Nitrobenzenes; C101520:P(2)-(1-(2-nitrophenyl)ethyl) ADP; D028044:Phosphate-Binding Proteins; D010725:Phosphocreatine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D003402:Creatine Kinase; D052281:Creatine Kinase, Mitochondrial Form; D008274:Magnesium; D003401:Creatine; D004229:Dithiothreitol