Arginine to citrulline replacement in substrates of phosphorylase kinase

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, trun cated 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 posit ion 16 and with two citrullines were phosphorylated less effectively than t he parent peptide; k(cat)/K-m values were approximately 20% the value with the parent peptide. Incorporation of the second citrulline had little chang e in the effectiveness of the peptide as a substrate although the kinetic p arameters with the citrulline peptides did show differences. The change in peptide phosphorylation did not seem to result from a change in peptide str ucture. 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 parame ters with the modified peptides seemed an effect of arginine replacement an d was likely a consequence of the loss of charge inasmuch as the size of ar ginine and citrulline are similar. Arginine 16 was concluded to be more imp ortant for phosphorylase kinase recognition than arginine-10. These finding s were consistent with the earlier studies using alanine replacement of arg inine in synthetic peptides as substrates for the holoenzyme form of phosph orylase kinase. (C) 2000 Elsevier Science B.V. All rights reserved.