Binding of acetyl-CoA to chicken liver pyruvate carboxylase.
Pyruvate carboxylase from chicken liver is a tetramer whose catalytic activity is completely dependent on the presence of acetyl-CoA. However, no direct evidence concerning the nature of the binding of the activator to the enzyme has been available. This is due in part to the instability of the enzyme and its ability to hydrolyze acetyl-CoA at an appreciable rate. The present studies on binding of the activator with the rapid flow dialysis method of Colowick and Womack ((1969) J. Biol. Chem. 244, 774-777) as modified by Klapper ((1970) Biochem. Biophys. Res. Commun. 38, 172-179) show four binding sites for acetyl-CoA. The binding dissociation constant at pH 7.2 is 13.9 muM as compared with an activation constant of 13.3 muM for the catalytic reaction at this pH. The relationship between acetyl-CoA concentration and catalytic activity is highly cooperative (nH = 2.9). The binding process also exhibits positive cooperativity but to a lower degree (nH = 1.9). Pyruvate carboxylase from chicken liver is rapidly inactivated and dissociated in the cold (0 degrees). The inactive protomeric form of the enzyme has been shown to be unable to bind acetyl-CoA at 0 degrees although the tetrameric species can do so. These results provide a plausible explanation for the catalytic inactivity of the protomer. The presence of acetyl-CoA results in an ultraviolet difference spectrum for the enzyme with a maximum at 280 nm. Half-maximal optical density difference is observed at an acetyl-CoA concentration of 9 muM, in reasonable agreement with the binding and activation constants.
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D003065:Coenzyme A / Q000031:analogs & derivatives; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D011485:Protein Binding; D011766:Pyruvate Carboxylase / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D046911:Macromolecular Substances; D000105:Acetyl Coenzyme A; D011766:Pyruvate Carboxylase; D003065:Coenzyme A