Substrate and inhibitor recognition by phosphorylase kinase and protein phosphatase-1

Abstract

Synthetic peptides based on residues 9 to 18 of glycogen phosphorylase were prepared containing citruUine at position 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). Both the mono-citruUine and the di-citruUine peptides were phosphorylated less effectively than the parent peptide; kcat/Km values were approximately 20% the value with the parent peptide. Incorporation of the second citrtdline had Little change in the effectiveness of the peptide as a substrate although the kinetic parameters with the citruUine peptides did show differences. The change in phosphorylation did not seem to result from a change in peptide structure. Two dimensional NMR studies of di-citruUine peptide are reported and show no change in the solution structure of the peptide compared to parent peptide. Thus, the change in kinetic parameters with the modified peptides seemed an effect of arginine replacement and was likely a 22 consequence of the loss of charge inasmuch as the size of arginine and citrulline are similar. The results also suggest that arginine-16 was more important for phosphorylase kinase recognition than arginine-10. This result was consistent with earlier studies using alanine to replace arginine in sjmthetic peptides as substrates for the holoenzyme form of phosphorylase kinase.