IODINE OXIDATION OF SULFHYDRYL GROUPS OF CREATINE KINASE

Evidence is presented for oxidation of the cysteinyl residues of creatine kinase to the level of sulfenate, for the reversal of this oxidation with mercaptans, and for the correlation of these changes with alterations in the specific binding and catalytic properties of the enzyme. In the presence of excess iodine, a total of 12 equiv of iodine/mole of creatine kinase was consumed in the oxidation of six sulfhydryl groups of this protein. The linear relationship found to exist between the loss of sulfhydryl groups and the consumption of iodine indicates that these six sulfhydryl groups were oxidized to the same extent, and that the reaction was specific for the oxidation of cysteinyl side chains. When [125I]I2 was used for the oxidation, the oxidized protein after gel filtration contained no significant amount of 125I. Thus, although the oxidation may proceed through a sulfenyl iodide intermediate, there is no indication of the formation of a stable protein sulfenyl iodide. Evidence obtained thus far is consistent with representation of the oxidation product as a sulfenic acid formed by hydrolysis of a sulfenyl iodide. The addition of 4 equiv of iodine/mole of enzyme leads to the oxidation of only two sulfhydryl groups, but results in the loss of all catalytic activity. The iodine-inactivated enzyme shows no changes in ultraviolet spectrum, and binds the substrate magnesium adenosine diphosphate, although not so strongly as the native protein. The inactive enzyme, prepared by the reaction with slightly less than 4 equiv of iodine/mole, can be reactivated by incubation with mercaptoethanol or dithiothreitol. This reactivation presumably occurs through the intermediate formation of a mixed disulfide with the subsequent regeneration of the original sulfhydryl group. It is apparent from the stoichiometry, the absence of iodine in the oxidized protein, and the reactivation with mercaptans, that the product of the oxidation of the sulfhydryl groups is a sulfenyl function, presumably the sulfenic acid group. This alteration of the SH groups in the active site produces little or no change in the physical properties measured, and yet is sufficient to abolish all enzymatic activity. © 1969, American Chemical Society. All rights reserved.