Global Kinetic Analysis of Mammalian E3 Reveals pH-dependent NAD+/NADH Regulation, Physiological Kinetic Reversibility, and Catalytic Optimum

Michael A. Moxley, Daniel A. Beard, Jason N. Bazil

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Mammalian E3 is an essential mitochondrial enzyme responsible for catalyzing the terminal reaction in the oxidative catabolism of several metabolites. E3 is a key regulator of metabolic fuel selection as a component of the pyruvate dehydrogenase complex (PDHc). E3 regulates PDHc activity by altering the affinity of pyruvate dehydrogenase kinase, an inhibitor of the enzyme complex, through changes in reduction and acetylation state of lipoamide moieties set by the NAD+/NADH ratio. Thus, an accurate kinetic model of E3 is needed to predict overall mammalian PDHc activity. Here, we have combined numerous literature data sets and new equilibrium spectroscopic experiments with a multitude of independently collected forward and reverse steady-state kinetic assays using pig heart E3. The latter kinetic assays demonstrate a pH-dependent transition of NAD+ activation to inhibition, shown here, to our knowledge, for the first time in a single consistent data set. Experimental data were analyzed to yield a thermodynamically constrained four-redox-state model of E3 that simulates pH-dependent activation/inhibition and active site redox states for various conditions. The developed model was used to determine substrate/product conditions that give maximal E3 rates and show that, due to non-Michaelis-Menten behavior, the maximal flux is different compared with the classically defined kcat.

Original languageEnglish (US)
Pages (from-to)2712-2730
Number of pages19
JournalJournal of Biological Chemistry
Volume291
Issue number6
DOIs
StatePublished - Feb 5 2016

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Pyruvate Dehydrogenase Complex
NAD
Kinetics
Oxidation-Reduction
Assays
Chemical activation
Acetylation
Enzyme Inhibitors
Metabolites
Catalytic Domain
Swine
Fluxes
Substrates
Enzymes
Experiments
Inhibition (Psychology)
Datasets

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Global Kinetic Analysis of Mammalian E3 Reveals pH-dependent NAD+/NADH Regulation, Physiological Kinetic Reversibility, and Catalytic Optimum. / Moxley, Michael A.; Beard, Daniel A.; Bazil, Jason N.

In: Journal of Biological Chemistry, Vol. 291, No. 6, 05.02.2016, p. 2712-2730.

Research output: Contribution to journalArticle

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