The activity of S-thiomethyl modified creatine kinase is due to the regeneration of free thiol at the active site.

Creatine kinase modified by S-methyl methanethiosulfonate and devoid of reactive thiol group has been reported to retain about 18-40% of the activity of the native enzyme. It has now been found that during the reaction catalyzed by the modified enzyme the rate increases with time and if the reaction is allowed to continue sufficiently long, the enzyme eventually recovers full activity. The presence of substrates is not required for the reactivation as suitable dilution after removal of MMTS in excess leads to complete reactivation of the MMTS modified enzyme with the simultaneous regeneration of reactive thiol per each dimeric molecule as shown by determinations with DTNB and IAN. The addition of MMTS during the course of reactivation again inactivates the reactivated enzyme. The activity recovery is therefore due to the regeneration of reactive thiol and it appears that the active-site thiols are essential for the activity of rabbit muscle creatine kinase.