EFFECT OF CITRULLINE FOR ARGININE REPLACEMENT ON THE STRUCTURE AND TURNOVER OF PHOSPHOPEPTIDE SUBSTRATES OF PROTEIN PHOSPHATASE-1

Phosphorylated and nonphosphorylated forms of a decapeptide correspond ing to residues 9 to 18 of glycogen phosphorylase were compared using two-dimensional nuclear magnetic resonance with assignment of both pep tides done by the sequential method. Both forms had little secondary s tructure, but there was evidence for an interaction between arginine-1 6 and phosphorylated serine at position 14, A change in the chemical s hift for the epsilon-nitrogen hydrogen of arginine in position 16 was observed in the spectrum of the phosphorylated peptide and was not evi dent in a phosphopeptide having citrulline in place of arginine-16, Hy drolysis catalyzed by protein phosphatase-l was decreased with the cit rulline-containing phosphopeptide compared to the arginine-containing phosphopeptide with effects observed on both k(cat) and K-m of the pho sphatase reaction. Alkaline phosphatase hydrolyzed these peptides and a di-citrulline peptide equally well, These results are consistent wit h arginine being favorable in the recognition of substrates by phospha tase-l, possibly recognition as an arginine-phosphoserine complex. As a model study, arginine and two analogs, citrulline and canavanine, we re examined for association with inorganic phosphate by nuclear magnet ic resonance spectrometry. P-31-NMR measurements showed that arginine and canavanine caused a shift in the phosphate resonance at 20 degrees C, Citrulline caused no change. Changes in chemical shift were measur ed over the pH range 5-9 with arginine and canavanine both causing a s light decrease in the apparent pK(a) of inorganic phosphate (Delta pK( a) approximate to 0.15). NaCl, NH4Cl, and guanidine hydrochloride show ed little effect on the resonance signal position of inorganic phospha te at pH 6.5, consistent with selectivity for the guanidino group. Tem perature (6 degrees, 20 degrees, and 37 degrees C) caused little chang e in the effect of arginine, but there was some dependency with canava nine, decreasing with temperature. Citrulline caused no change in the chemical shift of phosphate at any temperature. It was concluded that hydrogen bonded complexes were formed between the dianion of phosphate and the protonated form of arginine or canavanine with a bifurcated s tructure having preference for the omega-hydrogens. (C) 1998 Academic Press.