title: Arginine to citrulline replacement in substrates of phosphorylase kinase.
abstract: Synthetic peptides based on residues 9 to 18 of glycogen phosphorylase were prepared containing citrulline at position 10 or 16, or at both positions 10 and 16. The peptides were compared as substrates for a recombinant, truncated form of the catalytic subunit of phosphorylase kinase (residues 1-300). The peptide having citrulline at position 10 was phosphorylated the same as the parent peptide. Both the peptides with a single citrulline at position 16 and with two citrullines were phosphorylated less effectively than the parent peptide; k(cat)/K(m) values were approximately 20% the value with the parent peptide. Incorporation of the second citrulline had little change in the effectiveness of the peptide as a substrate although the kinetic parameters with the citrulline peptides did show differences. The change in peptide phosphorylation did not seem to result from a change in peptide structure. Two-dimensional nuclear magnetic resonance studies of di-citrulline peptide are reported and showed no change in the solution structure of the peptide compared to the parent peptide. Thus, the change in kinetic parameters with the modified peptides seemed an effect of arginine replacement and was likely a consequence of the loss of charge inasmuch as the size of arginine and citrulline are similar. Arginine-16 was concluded to be more important for phosphorylase kinase recognition than arginine-10. These findings were consistent with the earlier studies using alanine replacement of arginine in synthetic peptides as substrates for the holoenzyme form of phosphorylase kinase.
authors.affiliation: Signal Transduction Training Program, Iowa State University, Ames, IA 50011, USA.
authors.forename: C
authors.initials: C
authors.lastname: Bartleson
authors.forename: S
authors.initials: S
authors.lastname: Luo
authors.forename: D J
authors.initials: DJ
authors.lastname: Graves
authors.forename: B L
authors.initials: BL
authors.lastname: Martin
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D002956:Citrulline / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010764:Phosphorylase Kinase / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D002956:Citrulline; D001120:Arginine; D010764:Phosphorylase Kinase