ACETYL COENZYME-A CARBOXYLASE - RAPID PURIFICATION OF THE CHICK LIVER-ENZYME AND STEADY-STATE KINETIC-ANALYSIS OF THE CARBOXYLASE-CATALYZED REACTION

  • 1 January 1982
    • journal article
    • research article
    • Vol. 257  (2) , 924-929
Abstract
Avidin affinity chromatography was used to rapidly purify acetyl-CoA carboxylase to homogeneity in high yield from chicken liver. Dissociation of the purified carboxylase with dodecyl sulfate yielded a single size class of subunit polypeptide of 225,000 daltons. A steady state kinetic analysis of the carboxylase-catalyzed carboxylation of acetyl-CoA gave rise to intersecting line patterns in all double-reciprocal plots of initial velocity with each substrate pair, i.e., ATP .cntdot. Mg and HCO3- and acetyl-CoA. The kinetic mechanism involves a quaternary complex of the enzyme, ADP, Pi and acetyl-CoA rather than a double displacement as previously believed. The ordered addition of ATP, HCO3- and then acetyl-CoA, to the citrate-activated form of the carboxylase is the kinetic mechanism most consistent with the results.