[Role of the sulfhydryl groups of phosphorylase kinase]

The cysteinyl residues of rabbit skeletal muscle non-activated phosphorylase kinase were titrated by 5,5'-dithiobis (2-nitrobenzoic acid). According to their reactivity toward SH-reagent the accessible cysteinyl residues may be classified into three groups. Modification of the first group residues is accompanied by the enzyme activation. The inactivation is characterized by a biphasic kinetic profile. The fast and slow phases of inactivation correlate in time with modification of the second and third groups of cysteinyl residues. The apparent order of the reaction with respect to inhibitor concentrations for the slow and fast reactions gives a value of about 1, thus indicating that the enzyme contains two essential cysteinyl residues per protomer (alpha beta gamma delta). Mg-ATP and Mg-ADP protect the enzyme against inactivation and modification of sulfhydryl groups. It may be assumed that the two essential cysteinyl residues are located near the nucleotide-binding sites.