Evidence of a preferred kinetic pathway in the carnitine acetyltransferase reaction

Michael J. Kratochvil, Nick K. Balerud, Samantha J. Schindler, Michael A. Moxley

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


Mammalian carnitine acetyltransferase (CrAT) is a mitochondrial enzyme that catalyzes the reversible transfer of an acetyl group from acetyl-CoA to carnitine. CrAT knockout studies have shown that this enzyme is critical to sustain metabolic flexibility, or the ability to switch between different fuel types, an underlying theme of the metabolic syndrome. These recent physiological findings imply that CrAT dysfunction, or its catalytic impairment, may lead to disease. To gain insight into the CrAT kinetic mechanism, we conducted stopped-flow experiments in various enzyme substrate/product conditions and analyzed full progress curves by global fitting. Simultaneous mixing of both substrates with CrAT produced relatively fast kinetics that follows an ordered bi bi mechanism. A great preference for ordered binding is supported by stopped-flow double mixing experiments such that premixed CrAT with acetyl-CoA or CoA demonstrated a biphasic decrease in initial rate that produces about a 100-fold attenuation in catalysis. Double mixing experiments also revealed that the CrAT initial rate is inhibited by 50% in approximately 8 s by either acetyl-CoA or CoA premixing. Analysis of available CrAT structures support a substrate conformational change between acetyl-CoA/CoA binary versus ternary complexes. Additional viscosity-based kinetic experiments yielded strong evidence that product release is the rate limiting step in the CrAT-catalyzed reaction.

Original languageEnglish (US)
Article number108507
JournalArchives of Biochemistry and Biophysics
StatePublished - Sep 30 2020


  • Acetyl-CoA
  • Acetylcarnitine
  • Enzyme kinetics
  • Global fitting
  • Mitochondrial metabolism

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology


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