Comparison of activity and conformation changes during refolding of urea-denatured creatine kinase.

The course of the recovery of the enzymatic activity and the native conformation during the renaturation of urea-denatured creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) has been studied. Under suitable conditions, an activity recovery of 95% can be obtained and the reactivation follows a triphasic course. The initial two phases are relatively fast, whereas the slow phase takes some 24 h to reach completion. The recovery of the native conformation has been followed by changes in fluorescence, ultraviolet absorption and in exposed SH groups and has been shown to be a biphasic process. Both the reactivation and the refolding processes are independent of protein concentrations within a certain range, showing that the dimerization of the enzyme molecule is not rate-limiting. A comparison of the rate constants for the refolding of the molecule with those for the recovery of its catalytic activity shows that these are not synchronized and the activity recovery approaches completion after the refolding and dimerization of the subunits so far as can be detected by the methods employed. The final stage of refolding with complete activity recovery probably involves subtle conformational changes of the dimeric enzyme molecule not detectable by the physiochemical methods used in the present study.