The number of catalytic sites in creatine phosphokinase as determined by a study of its reactive sulphydryl groups

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Abstract
Creatine phosphokinase reacts rapidly with iodoacetate to produce 2 equiv. of iodide as determined with the iodide electrode. Complete enzyme inactivation results and is associated with the alkylation of 2 SH groups. The possibility of one catalytic site with 2 SH groups essential for activity is excluded. With one sample of creatine phosphokinase the substrate equilibrium mixture protects one SH group but not the other, This indicates one catalytic site containing one essential SH group. This view is supported by the reaction of creatine phosphokinase with p-nitrophenylacetate in which the catalytic site SH group is selectively inhibited as judged by the subsequent reaction with iodoacetate. The SH groups of a second sample of creatine phosphokinase are equally affected by the substrate equilibrium mixture and by p-nitrophenylacetate. This suggests 2 catalytic sites with one SH group at each for this preparation. The possibilities that the differences in the 2 preparations are of genetic origin or result from the isolation procedure are discussed.