Characterization of recombinant human acetyl-CoA carboxylase-2 steady-state kinetics |
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Authors: | Virendar K. Kaushik Michael Kavana Jessica M. Volz Stephen C. Weldon Susan Hanrahan Jian Xu Shari L. Caplan Brian K. Hubbard |
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Affiliation: | 1. Cardiovascular and Metabolism Disease Area, Novartis Institutes for BioMedical Research, Inc., Cambridge, Massachusetts, USA;2. Tufts University, Friedman School of Nutrition Science and Policy, USDA-HNRCA, Boston, Massachusetts, USA |
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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 (HCO3?), 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 kcat and kcat/KM for ATP and acetyl-CoA. |
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