Phosphorus-31 nuclear magnetic resonance study of the active site phosphohistidine and regulatory phosphoserine residues of rat liver ATP-citrate lyase.
31P NMR has been used to investigate the nature of the two chemically distinct phosphorylation sites of ATP-citrate lyase from rat liver. The "regulatory" or "structural" phosphorylation site is acid stable and known to be phosphoserine. The "catalytic" site is very acid labile and has been suggested by different workers to contain either phosphohistidine or an acyl phosphate group. We have demonstrated the presence of both endogenous phosphoserine and phosphoserine introduced after treatment of the lyase with the catalytic subunit of cAMP-dependent protein kinase. This structural phosphate group could be titrated and was readily removed by alkaline phosphatase; these facts, together with the narrow line width of the 31P NMR signal, suggest that it is relatively mobile and located near the surface of the protein. 31P NMR spectra of ATP-citrate lyase that had previously been exposed to fairly high concentrations of potassium chloride (1.5 M), or that had been denatured in detergent and 2-mercaptoethanol, clearly identified phosphohistidine as the catalytic phosphate group. That phosphohistidine is indeed a catalytic intermediate was demonstrated by the disappearance of the resonance in the presence of the substrates citrate and coenzyme A. The line width of the phosphohistidine resonance indicated that the catalytic phosphohistidine residue has negligible residual mobility on the protein. These results are consistent with the pattern of earlier observations on the chemical environments of phospho groups that serve a regulatory or structural role as opposed to a catalytic function in proteins.
D001275:ATP Citrate (pro-S)-Lyase / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D006639:Histidine / Q000031:analogs & derivatives* / Q000032:analysis; D008099:Liver / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008297:Male; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis*; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012694:Serine / Q000031:analogs & derivatives*
D010768:Phosphoserine; D012694:Serine; D006639:Histidine; D001275:ATP Citrate (pro-S)-Lyase; D011494:Protein Kinases; C005436:phosphohistidine