The Sulfhydryl Groups of Muscle Phosphorylase: IV. REACTIVITIES AS RELATED TO CHANGES IN PROTEIN STRUCTURE

Abstract

Abstract Reaction of muscle phosphorylase with 14C-iodoacetamide followed by determination of radioactivity in pepsin-derived peptides permits the measurements of rates of alkylation of cysteinyl peptides of known amino acid sequence. The two —SH groups of Peptides B1 and B2, previously shown not to be related to the structure and function of this enzyme, were not affected significantly by the experiments reported in this paper. Two —SH groups per monomer of phosphorylase a, contained in Peptides N and A, are related to structure and function because, as reported before, their alkylation results in a dissociated, inactive protein. The presence of AMP results in no alkylation of Peptide A and no dissociation into monomers, so that the loss of activity is correlated with the alkylation of the single —SH group of peptide N in each monomer, and both processes exhibit the same rate. Imidazole citrate buffer causes phosphorylase b to dissociate into monomers, and in this case also the —SH group of Peptide A is not alkylated and the rate of inactivation is the same as the rate of alkylation of the single —SH group of Peptide N. The rate of alkylation of the N peptide of the monomer is more than 100 times that of the dimer, is similar to the rate for model compounds, and suggests that in this monomer the —SH group is freely exposed on the surface. AMP, which maintains phosphorylase b in an oligomeric form in imidazole citrate, lowers the rate of alkylation and inactivation some 6-fold. The reduction of the aldimine linkage between pyridoxal-5-P and the enzyme does not prevent phosphorylase b from dissociating in imidazole citrate, and, as with the native enzyme, Peptide A is not alkylated, while the inactivation is correlated with the reaction of the N peptide. In contrast to the monomer formed in imidazole citrate, the monomeric apoenzyme formed by the removal of pyridoxal-5-P from phosphorylase b becomes alkylated in both its A and N peptides when treated with iodoacetamide. Phosphorylase a does not dissociate in imidazole citrate, and this buffer causes only a moderate increase in the rate of inactivation. The reaction of iodoacetamide with phosphorylase b in imidazole citrate causes a change in the spectrum of the pyridoxal-5-P which is indicative of the formation of a Schiff base. The rates of inactivation, of alkylation of the N peptide, and of the Schiff base formation are equivalent. The results are discussed in relation to the interdependence of diverse sites on the phosphorylase molecule. The various chemical modifications of specific sites, or the binding of ligands to their sites, result in changes transmitted through the protein structure as reflected by the altered character of other sites. In particular, the reactivities of the —SH groups of Peptides N and A, as measured by the rates of reaction with iodoacetamide, are altered in a definable manner by treatments of the other sites, and each variation in reactivity may represent a conformational change in the protein.