Mechanistic studies of the long chain acyl-CoA synthetase Faa1p from Saccharomyces cerevisiae

Hong Li, Elaina M. Melton, Steven Quackenbush, Concetta C. DiRusso, Paul N. Black

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Long chain acyl-CoA synthetase (ACSL; fatty acid CoA ligase: AMP forming; EC 6.2.1.3) catalyzes the formation of acyl-CoA through a process, which requires fatty acid, ATP and coenzymeA as substrates. In the yeast Saccharomyces cerevisiae the principal ACSL is Faa1p (encoded by the FAA1 gene). The preferred substrates for this enzyme are cis-monounsaturated long chain fatty acids. Our previous work has shown Faa1p is a principal component of a fatty acid transport/activation complex that also includes the fatty acid transport protein Fat1p. In the present work hexameric histidine tagged Faa1p was purified to homogeneity through a two-step process in the presence of 0.1% η-dodecyl-β-maltoside following expression at 15 °C in Escherichia coli. In order to further define the role of this enzyme in fatty acid transport-coupled activation (vectorial acylation), initial velocity kinetic studies were completed to define the kinetic parameters of Faa1p in response to the different substrates and to define mechanism. These studies showed Faa1p had a Vmax of 158.2 nmol/min/mg protein and a Km of 71.1 μM oleate. When the concentration of oleate was held constant at 50 μM, the Km for CoA and ATP were 18.3 μM and 51.6 μM respectively. These initial velocity studies demonstrated the enzyme mechanism for Faa1p was Bi Uni Uni Bi Ping Pong.

Original languageEnglish (US)
Pages (from-to)1246-1253
Number of pages8
JournalBiochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
Volume1771
Issue number9
DOIs
StatePublished - Sep 1 2007

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Coenzyme A Ligases
Acyl Coenzyme A
Saccharomyces cerevisiae
Fatty Acids
Coenzyme A
Oleic Acid
Fatty Acid Transport Proteins
Enzymes
Adenosine Triphosphate
Acylation
Adenosine Monophosphate
Ligases
Histidine
Yeasts
Escherichia coli
Genes
Proteins

Keywords

  • Fatty acid transport
  • Long chain acyl CoA synthetase
  • Mechanistic enzymology

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

Cite this

Mechanistic studies of the long chain acyl-CoA synthetase Faa1p from Saccharomyces cerevisiae. / Li, Hong; Melton, Elaina M.; Quackenbush, Steven; DiRusso, Concetta C.; Black, Paul N.

In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, Vol. 1771, No. 9, 01.09.2007, p. 1246-1253.

Research output: Contribution to journalArticle

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