BINDING OF ACETYL-COA TO CHICKEN LIVER PYRUVATE-CARBOXYLASE

  • 1 January 1977
    • journal article
    • research article
    • Vol. 252  (1) , 51-56
Abstract
Pyruvate carboxylase [EC 6.4.1.1] from chicken liver is a tetramer whose catalytic activity is completely dependent on the presence of acetyl-CoA. The present studies on binding of the activator with the rapid flow dialysis method of Colowick and Womack as modified by Klapper show 4 binding sites for acetyl-CoA. The binding Kd at pH 7.2 is 13.9 .mu.M as compared with an activation constant of 13.3 .mu.M 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.degree.). The inactive protomeric form of the enzyme is unable to bind acetyl-CoA at 0.degree. 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 UV 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 .mu.M, in reasonable agreement with the binding and activation constants.