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 i ts protection by substrates during guanidine denaturation were investi gated by monitoring the ESR spectra of the nitroxide radical covalentl y bound to the reactive thiols of the enzyme. For the enzyme undenatur ed (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 increasi ng denaturant concentration. At concentrations of guanidine hydrochlor ide higher than 1 M, the structure of the enzyme molecule is monomorph ic and becomes looser and looser with an increase of guanidine hydroch loride concentration. The existence of a nucleotide substrate complex protects the structure at the active sites of the enzyme from being ch anged, up to a concentration of denaturant of 0.2 M, while creatine ha s no protective effect.