title: Phosphorylation of recombinant human ATP:citrate lyase by cAMP-dependent protein kinase abolishes homotropic allosteric regulation of the enzyme by citrate and increases the enzyme activity. Allosteric activation of ATP:citrate lyase by phosphorylated sugars.
abstract: Recombinantly expressed human ATP:citrate lyase was purified from E. coli, and its kinetic behavior was characterized before and after phosphorylation. Cyclic AMP-dependent protein kinase catalyzed the incorporation of only 1 mol of phosphate per mole of enzyme homotetramer, and glycogen synthase kinase-3 incorporated an additional 2 mol of phosphate into the phosphorylated protein. Isoelectric focusing revealed that all of the phosphates were incorporated into only one of the four enzyme subunits. Phosphorylation resulted in a 6-fold increase in V(max) and the conversion of citrate dependence from sigmoidal, displaying negative cooperativity, to hyperbolic. The phosphorylated recombinant enzyme is more similar to the enzyme isolated from mammalian tissues than unphosphorylated enzyme with respect to the K(m) for citrate, CoA, and ATP, and the specific activity. Fructose 6-phosphate was found to be a potent activator (60-fold) of the unphosphorylated recombinant enzyme, with half-maximal activation at 0.16 mM, which results in a decrease in the apparent K(m) for citrate and ATP, as well as an increase in the V(max) of the reaction. Thus, human ATP:citrate lyase activity is regulated in vitro allosterically by phosphorylated sugars as well as covalently by phosphorylation.
authors.affiliation: Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794-8661, USA.
authors.forename: I A
authors.initials: IA
authors.lastname: Potapova
authors.forename: M R
authors.initials: MR
authors.lastname: El-Maghrabi
authors.forename: S V
authors.initials: SV
authors.lastname: Doronin
authors.forename: W B
authors.initials: WB
authors.lastname: Benjamin
mesh_terms: D001275:ATP Citrate (pro-S)-Lyase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000494:Allosteric Regulation; D000818:Animals; D002384:Catalysis; D019343:Citric Acid / Q000737:chemistry / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*
chemical_list: D011994:Recombinant Proteins; D013403:Sugar Phosphates; D019343:Citric Acid; D001275:ATP Citrate (pro-S)-Lyase; D017868:Cyclic AMP-Dependent Protein Kinases