Redox control of catalysis in ATP-citrate lysate from rat liver.
In thiol redox buffers at pH 8.0, rat liver ATP-citrate lyase is in equilibrium between an oxidised inactive form and a reduced active form. The reduced enzyme is inactivated by oxidised glutathione (GSSG) at a rate of 45 min-1.M-1 and the oxidised enzyme is activated by reduced glutathione (GSH) at a rate of 3.2 min-1.M-1. At redox equilibrium, the enzyme activity depends on the ratio [GSH]2/[GSSG]. The inactivation involves formation of a protein-protein disulphide rather than a protein-glutathione complex. This reaction has Keq = 78 +/- 7 mM for the oxidative reaction. Activity can therefore be controlled by the redox state of the cell, being more active in the fed state than in the oxidatively stressed state. This redox process is also important in the in vitro enzyme assay, where ATP-citrate lyase is in redox equilibrium with oxygen and either dithiothreitol or 2-mercaptoethanol. Reduction is a two-step process, requiring high concentrations of reductant for full activation (30 mM dithiothreitol or 200 mM 2-mercaptoethanol). The enzyme inhibitor, Medica-16 raises the redox equilibrium constant to greater than 400 mM. It binds more tightly to the oxidised form of the enzyme, with Ki less than 40 microM compared to 180 microM for the reduced form.
Wells
T N
TN
Molecular Enzymology Laboratory, Smith Kline Beecham, The Frythe, Welwyn, England.
Saxty
B A
BA
D001275:ATP Citrate (pro-S)-Lyase / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005978:Glutathione / Q000494:pharmacology; D000960:Hypolipidemic Agents / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008623:Mercaptoethanol / Q000494:pharmacology; D010084:Oxidation-Reduction; D010169:Palmitic Acids / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D000960:Hypolipidemic Agents; D010169:Palmitic Acids; D008623:Mercaptoethanol; C056166:MEDICA 16; D001275:ATP Citrate (pro-S)-Lyase; D005978:Glutathione; D004229:Dithiothreitol