The sulfhydryl groups of muscle phosphorylase. 3. Identification of cysteinyl peptides related to function.

Iodoacetamide inactivates glycogen phosphorylase and causes it to dissociate into monomers of 92,500 molecular weight. Three -SH groups on each monomer are alkylated, but the first reacts as rapidly as do model compounds and its alkylation does not cause inactivation. The remaining two -SH groups are alkylated several hundred times slower, at rates comparable to the loss of activity. The three --SH groups were labeled with ‘*CLiodoacetamide. The radioactive peptides were isolated from pepsin digests and characterized sufficiently to identify them with certain of the cysteinyl peptides previously isolated and sequenced. The -SH group whose alkylation does not affect activity has been tentatively assigned to the sequence GlyCys-Arg-Asp (peptide B). The sequences of the other two cysteinyl peptides which are alkylated concomitantly with the loss of activity are Ala-Cys-Ala-Phe (peptide N) and Asn-Ala-Cys-Asp (peptide A). Another six -SH groups per monomer are not readily alkylated in the native enzyme. Paper electrophoresis of pepsin digests of the W-carboxamidomethyl peptides permits one to compare the kinetics of alkylation of the various peptides with the rates of inactivation. For phosphorylase b, the rate of alkylation of the N peptide is similar to the rate of inactivation and suggests that the alkylation of only this peptide causes loss of activity and dissociation to monomers. Peptide A is alkylated at 50% of the rate of peptide N. For phosphorylase a, the sum of the rates of alkylation of both peptides is similar to the rate of inactivation, and suggests that the alkylation of either results in an inactive enzyme. Alternative interpretations are possible, however, and are discussed. The presence of AMP prevents the alkylation of the peptide A of phosphorylase a, so that in this circumstance the activity loss can be assigned solely to the alkylation of the -SH group of peptide N.