Chemical modification of phosphorylase b by tetranitromethane. Identification of a functional tyrosyl residue.
Tetranitromethane, C(NO2)4, a reagent for tyrosyl residues, was found to inactivate irreversibly rabbit skeletal muscle glycogen phosphorylase b. Under the chosen conditions seven tyrosyl residues, namely Tyr-75, 203, 262, 280, 403, 552 and 647, were found to be nitrated. Inactivation was prevented by the presence of the allosteric activator 5'-AMP during nitration. Under these latter conditions one of the reactive tyrosyl residues was not modified by C(NO2)4; thus, this residue appeared to be essential for either catalytic activity or allosteric activation. Tryptic digests of phosphorylase b, reacted with C(NO2)4 in the absence and presence of 5'AMP, were fractionated by gel filtration. The peptide mixtures were further purified by reverse-phase HPLC. One of the peptides contained the tyrosyl residue which was modified by C(NO2)4 only in the absence of 5'AMP. The sequence of this peptide was determined. The amino acid residue which is responsible for the loss of activity upon reaction with C(NO2)4 was identified in the amino acid sequence of phosphorylase b as tyrosine-75. Of the other residues modified in the presence and in the absence of C(NO2)4, tyrosine-403 contributes to the glycogen-storage site whereas Tyr-280 is close to the alpha-D-glucose-binding site. These residues, exposed to the solvent both in the presence and in the absence of 5'AMP, are not essential for catalytic activity.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002384:Catalysis; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D008697:Methane / Q000031:analogs & derivatives*; D009132:Muscles / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D010763:Phosphorylase b / Q000037:antagonists & inhibitors*; D006005:Phosphorylases / Q000037:antagonists & inhibitors*; D011817:Rabbits; D013774:Tetranitromethane / Q000494:pharmacology*; D014443:Tyrosine / Q000032:analysis*
D000596:Amino Acids; D010446:Peptide Fragments; D014443:Tyrosine; D010763:Phosphorylase b; D006005:Phosphorylases; D013774:Tetranitromethane; D008697:Methane