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 corresponding to residues 9 to 18 of glycogen phosphorylase were compared using two-dimensional nuclear magnetic resonance with assignment of both peptides done by the sequential method. Both forms had little secondary structure, but there was evidence for an interaction between arginine-16 and phosphorylated serine at position 14. A change in the chemical shift for the epsilon-nitrogen hydrogen of arginine in position 16 was observed in the spectrum of the phosphorylated peptide and was not evident in a phosphopeptide having citrulline in place of arginine-16. Hydrolysis catalyzed by protein phosphatase-1 was decreased with the citrulline-containing phosphopeptide compared to the arginine-containing phosphopeptide with effects observed on both kcat and Km of the phosphatase reaction. Alkaline phosphatase hydrolyzed these peptides and a di-citrulline peptide equally well. These results are consistent with arginine being favorable in the recognition of substrates by phosphatase-1, possibly recognition as an arginine-phosphoserine complex. As a model study, arginine and two analogs, citrulline and canavanine, were examined for association with inorganic phosphate by nuclear magnetic resonance spectrometry. 31P-NMR measurements showed that arginine and canavanine caused a shift in the phosphate resonance at 20 degreesC. Citrulline caused no change. Changes in chemical shift were measured over the pH range 5-9 with arginine and canavanine both causing a slight decrease in the apparent pKa of inorganic phosphate (DeltapKa approximately 0.15). NaCl, NH4Cl, and guanidine hydrochloride showed little effect on the resonance signal position of inorganic phosphate at pH 6.5, consistent with selectivity for the guanidino group. Temperature (6 degrees, 20 degrees, and 37 degreesC) caused little change in the effect of arginine, but there was some dependency with canavanine, 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 structure having preference for the omega-hydrogens.
Martin
B L
BL
Department of Biochemistry, University of Tennessee, Memphis, Tennessee, 38163, USA. bmartin@utmem1.utmem.edu
Luo
S
S
Kintanar
A
A
Chen
M
M
Graves
D J
DJ
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000737:chemistry*; D001665:Binding Sites; D002172:Canavanine / Q000737:chemistry; D002956:Citrulline / Q000737:chemistry*; D004789:Enzyme Activation; D006868:Hydrolysis / Q000187:drug effects; D009842:Oligopeptides / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry*; D010749:Phosphoprotein Phosphatases / Q000737:chemistry*; D006005:Phosphorylases / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D054645:Protein Phosphatase 1; D011817:Rabbits; D013379:Substrate Specificity
D009842:Oligopeptides; D010710:Phosphates; D010748:Phosphopeptides; D002956:Citrulline; D002172:Canavanine; D001120:Arginine; D006005:Phosphorylases; D000469:Alkaline Phosphatase; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1