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 Faalp (encoded by the FAA1 gene). The preferred substrates for this enzyme are cis-monounsaturated long chain fatty acids. Our previous work has shown Faalp 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 Faalp was purified to homogeneity through a two-step process in the presence of 0.1% eta-dodecyl-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 Faalp in response to the different substrates and to define mechanism. These studies showed Faalp had a V-max of 158.2 nmol/min/mg protein and a K-m of 71.1 mu M oleate. When the concentration of oleate was held constant at 50 mu M, the K-m for CoA and ATP were 18.3 mu M and 51.6 mu M respectively. These initial velocity studies demonstrated the enzyme mechanism for Faalp was Bi Uni Uni Bi Ping Pong. (C) 2007 Elsevier B.V. All rights reserved.