Phosphorus-31 nuclear magnetic resonance study of the active site phosphohistidine and regulatory phosphoserine residues of rat liver ATP-citrate lyase

Abstract

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.

Phosphorylation by cyclic AMP‐dependent protein kinase does not affect the association of ATP citrate‐lyase with isolated mitochondria

“…1 shows the rate and extent of incorporation of phosphate from ATP into the structural site of ATP citrate-lyase, as catalyzed by the catalytic subunit of cyclic AMP-dependent protein kinase. The incorporation reaches a plateau near a level of two phosphoryl groups per tetramer, a value that is consistent with an earlier suggestion [1] of halfsites reactivity for phosphorylation of this enzyme. Thus, the phosphorylated enzyme that was used routinely in this study was obtained after 90 min of incubation under these conditions, when the incorporation is near maximal.…”

“…ATP citrate-lyase was purified to homogeneity from livers of male Sprague-Dawley rats that had been starved for 2 days and then refed a high carbohydrate diet for 3 days immediately prior to killing, essentially as in [1,17]. Enzyme activity was determined by the coupled assay outlined by Osterlund and Bridger [18].…”

“…Any 32p incorporated at the catalytic site was removed by incubation with citrate and CoA [1], and the phosphorylated lyase was isolated by gel filtration on Sephadex G-100. Preparations of ATP citratelyase in which the phosphoryl groups were removed from the structural sites were obtained by incubation of the phosphorylated enzyme with 0.75 U Escherichia coli alkaline phosphatase in 50 mM Tris-HC1 (pH 8.4), 1 mM dithiothreitol for 6 h at 30°C.…”

“…One of these is the result of formation of a phospho-enzyme intermediate in catalysis, through the transient formation of a phosphohistidine residue at the active site [1]. In addition, the enzyme may be phosphorylated at a serine residue at the 'regulatory' or structural site in vivo [2] or by the action of cyclic AMP-dependent [1,3,4] and -independent [5] protein kinases in vitro, and in response to insulin [6,7] and glucagon [4,8,9]. Curiously, phosphorylation at the structural site in Correspondence address: W.A.…”

Advances in development of new tools for the study of phosphohistidine

“…Similarly, Williams et al 76 , observed only τ-pHis by 31 ATP phosphorylated rat liver NDPK, only π-pHis was detected in the hydrolysate by TLC analysis against the reference compound. In the same study, only τ-pHis was detected by TLC from the base hydrolysate of ( 32 P) phosphorylated rat adrenal ACLY.…”