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 . Mg and HCO3(-) and acetyl-CoA. It was concluded that 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.
Highlights
Avidin affinity chromatographywas used to rapidly mechanism [1, 8], i.e. a double displacement kinetic mechapurify acetyl-coA carboxylase to homogeneity inhigh nism
We show by steady state kinetic analysisthattheacetyl-coA carboxylase-catalyzed reaction does not proceed by a double displacement mechanism as previously believed
The results presented are most consistent with an ordered Ter Terkinetic mechanism
Summary
From the Departmentof Physiological Chemistry, The Johns HopkinUs niversity School of Medicine, Baltimore, Maryland21205. Each assay was initiated by the addition of 7 pg of purified acetyl-coA carboxylase (4.5 to 5.5 units/mg of protein) to 1. Evidence implicating these two "half-reactions'' in the overall ml of reaction mixture at 37 "C containing 10 mM potassium citrate, process catalyzed by the mammalian and aviancarboxylases derives both from isotopic-exchange studies [1,2,3,4] and direct stoichiometric experiments [1, 5] withenzyme The pellet is gently resuspended with the aid of a Teflon homogenizer in 1 liter of a buffer containing 144 g of ammonium sulfate(25% saturated), 0.05 M potassium phosphate, pH7.0,0.5mM EDTA, 5 mM
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