Mechanistic studies of the long chain acyl-CoA synthetase Faa1p from Saccharomyces cerevisiae.
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% eta-dodecyl-beta-maltoside following expression at 15 degrees 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 microM oleate. When the concentration of oleate was held constant at 50 microM, the Km for CoA and ATP were 18.3 microM and 51.6 microM respectively. These initial velocity studies demonstrated the enzyme mechanism for Faa1p was Bi Uni Uni Bi Ping Pong.
Li
Hong
H
Center for Metabolic Disease, Ordway Research Institute, Albany, NY 12208, USA.
Melton
Elaina M
EM
Quackenbush
Steven
S
DiRusso
Concetta C
CC
Black
Paul N
PN
D000255:Adenosine Triphosphate / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019301:Oleic Acid / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D029701:Saccharomyces cerevisiae Proteins; D019301:Oleic Acid; D000255:Adenosine Triphosphate; D003066:Coenzyme A Ligases; C511244:Faa1 protein, S cerevisiae