Creatine kinase: The characteristics of the enzyme regenerated from the thio-methylated creatine kinase reflect a differentiation in function between the two reactive thiols

Abstract

The activity of S-thiomethyl-modified creatine kinase is due to regeneration of the free thiol (Hou and Vollmer, Biochim Biophys Acta (1994) 1205, 83–88). Characteristics of enzyme regenerated from the S-thiomethyl-modified creatine kinase are reported in the present study. The intrinsic fluorescence of the regenerated enzyme is similar to that of the native enzyme in the presence or absence of the dead-end complex. Regenerated CK (rCK) with full activity has only one reactive thiol. The rate constant of the rCK-reactive thiol reacting with DTNB is close to that of the slow phase of the reactive thiols of the native enzyme. If the IAM-modified rCK is treated with the same method as that for obtaining the rCK, the thiol-methylated reactive thiol of the rCK is reduced to a free SH and a regenerated enzyme, RCK, is produced with about 10% of the rCK activity. Therefore, the different roles of the two reactive thiols of creatine kinase may stem from the characteristics of the rCK, which suggests that only one of the two reactive thiols is related to the activity of the enzyme and the slower phase thiol (the first SH) in the modification reaction with DTNB is directly related to the enzymatic activity while the faster phase thiol (the second SH) assists the first SH. This compensatory mechanism is proposed in the present study to interpret the dispute on the reactive SH role in the enzymatic catalysis.