Spin-labeling probe on conformational change at the active sites of creatine kinase during denaturation by guanidine hydrochloride.
The conformational change at the active sites of creatine kinase and its protection by substrates during guanidine denaturation were investigated by monitoring the ESR spectra of the nitroxide radical covalently bound to the reactive thiols of the enzyme. For the enzyme undenatured (pH 9.0) and in the presence of low concentrations of guanidine, i.e. less than 1 M, there are two kinds of enzyme molecule, one of which is bearing a compact structure at the active site and the other is of a looser structure. The content of the latter increases with increasing denaturant concentration. At concentrations of guanidine hydrochloride higher than 1 M, the structure of the enzyme molecule is monomorphic and becomes looser and looser with an increase of guanidine hydrochloride concentration. The existence of a nucleotide substrate complex protects the structure at the active sites of the enzyme from being changed, up to a concentration of denaturant of 0.2 M, while creatine has no protective effect.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D001665:Binding Sites; D002845:Chromatography; D003401:Creatine / Q000378:metabolism / Q000494:pharmacology; D003402:Creatine Kinase / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000031:analogs & derivatives; D015335:Molecular Probes / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013113:Spin Labels
D006146:Guanidines; D015335:Molecular Probes; D013113:Spin Labels; D000244:Adenosine Diphosphate; D003402:Creatine Kinase; D019791:Guanidine; D003401:Creatine; D007460:Iodoacetamide