Characterization of recombinant human acetyl-CoA carboxylase-2 steady-state kinetics.
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.
Kaushik
Virendar K
VK
Novartis Institutes for BioMedical Research, Inc., Cambridge, Massachusetts, USA. virendar.kaushik@novartis.com
Kavana
Michael
M
Volz
Jessica M
JM
Weldon
Stephen C
SC
Hanrahan
Susan
S
Xu
Jian
J
Caplan
Shari L
SL
Hubbard
Brian K
BK
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
D011994:Recombinant Proteins; C520105:ACACB protein, human; D000103:Acetyl-CoA Carboxylase