title: Characterization of recombinant human acetyl-CoA carboxylase-2 steady-state kinetics.
abstract: Acetyl-CoA carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, a key metabolite in the fatty acid synthetic and oxidation pathways. The present study describes the steady-state kinetic analysis of a purified recombinant human form of the enzyme, namely ACC2, using a novel LC/MS/MS assay to directly measure malonyl-CoA formation. Four dimensional matrices, in which bicarbonate (HCO(3)(-)), ATP, acetyl-CoA, and citrate were varied, and global data fitting to appropriate steady-state equations were used to generate kinetic constants. Product inhibition studies support the notion that the enzyme proceeds through a hybrid (two-site) random Ter Ter mechanism, one that likely involves a two-step reaction at the biotin carboxylase domain. Citrate, a known activator of animal forms of ACC, activates both by increasing k(cat) and k(cat)/K(M) for ATP and acetyl-CoA.
authors.affiliation: Novartis Institutes for BioMedical Research, Inc., Cambridge, Massachusetts, USA. virendar.kaushik@novartis.com
authors.forename: Virendar K
authors.initials: VK
authors.lastname: Kaushik
authors.forename: Michael
authors.initials: M
authors.lastname: Kavana
authors.forename: Jessica M
authors.initials: JM
authors.lastname: Volz
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Weldon
authors.forename: Susan
authors.initials: S
authors.lastname: Hanrahan
authors.forename: Jian
authors.initials: J
authors.lastname: Xu
authors.forename: Shari L
authors.initials: SL
authors.lastname: Caplan
authors.forename: Brian K
authors.initials: BK
authors.lastname: Hubbard
mesh_terms: D000103:Acetyl-CoA Carboxylase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D002853:Chromatography, Liquid; D006801:Humans; D007700:Kinetics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D053719:Tandem Mass Spectrometry
chemical_list: D011994:Recombinant Proteins; C520105:ACACB protein, human; D000103:Acetyl-CoA Carboxylase