Conformational changes of active sites during refolding of urea-denatured creatine kinase

The course of the recovery of the active site conformation during refolding of urea-denatured creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) has been studied. The recovery of the active site conformation has been followed by changes in probe fluorescence of active site of the enzyme labeled by o-phthaldehyde and has been shown to be a triphasic process. A comparison of the rate constants for the conformational recovery of the active site with those for the recovery of enzyme catalytic activity shows that these are synchronized. The results obtained show clearly that, although recovery of partial activity and conformation of the active site occur in reactions with nearly the same rate as those of the refolding processes observed, the complete recovery of the enzyme activity and the active site conformation can only be obtained long after any detectable conformational change of whole enzyme molecule.