Mechanistic Studies of the Long Chain Acyl-CoA Synthetase Faa1p from Saccharomyces cerevisiae

Authors

Hong Li, Center for Metabolic Disease, Ordway Research Institute
Elaina M. Melton, Center for Metabolic Disease, Ordway Research Institute
Steven Quackenbush, Center for Metabolic Disease, Ordway Research Institute
Concetta C. DiRusso, Center for Metabolic Disease, Ordway Research InstituteFollow
Paul N. Black, Center for Metabolic Disease, Ordway Research InstituteFollow

Document Type

Article

Date of this Version

2007

Citation

Biochim Biophys Acta. 2007 September ; 1771(9): 1246–1253.

Comments

Copyright © 2007 Elsevier B.V.

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 V_max of 158.2 nmol/ min/mg protein and a K_m of 71.1μM oleate. When the concentration of oleate was held constant at 50μM, the K_m for CoA and ATP were 18.3μM and 51.6μM respectively. These initial velocity studies demonstrated the enzyme mechanism for Faa1p was Bi Uni Uni Bi Ping Pong.