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

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% 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.

Figure 1. Purification of the long chain acyl-CoA synthetase Faa1p following expression in E. coli

A. SDSPAGE of protein during the different steps of purification (S200-1 and S200-2 represents combined and concentrated fractions from the Faa1p peak from the S200 column); MW – molecular weight standards (indicated on the left). B. Determination of the native molecular weight of purified Faa1p in 50mM Hepes, pH7.4; 1mM ATP, 5mM MgCl₂, 0.5M NaCl, 10% glycerol, 0.05% DDM using an analytical size exclusion column (Superose™ 6 10/300 GL). The insert shows the elution position of Faa1p at ~17ml (arrow) relative to the standards used to calibrate the column (bovine thyroglobulin (670,000), bovine gamma globulin (158,000), chicken ovalbumin (44,000), horse myoglobin (17,000) and vitamin B-12 (1,350).

Figure 2. Characteristics of purified Faa1p

A. Acyl-CoA synthetase activity as a function of enzyme concentration; insert illustrates the linearity of the reaction between 0.5 and 3µg of enzyme. B. Lineweaver-Burke plot of the enzyme reaction as a function of varying oleate (C_18:1) concentration (5–200µM) using 1.5µg of purified enzyme. ATP and CoA concentrations were held constant at 10mM and 200µM respectively. V_o⁻¹ values are (nmol/min/mg protein)⁻¹.

Figure 3. Activity and stability of purified Faa1p

A. Enzyme activity was measured over a range of pH values using 1.5µg purified Faa1p; the activity at pH of 8.0 was set to 100% (163.7±12.9 nmol/min/mg protein). B. The thermal stability of Faa1p (1.5µg) was determined following incubation at the noted temperatures for 30min at which time activity was measured; the activity of Faa1p when maintained at 30°C was set to 100% (157.5±17.3 nmol/min/mg protein). The error bars are the standard error of the mean (n=3).

Figure 4. Initial velocity studies of the acyl-CoA synthetase reaction using 1.5µg purified Faa1p

Double reciprocal plots where A. CoA concentrations were held constant at 200µM and ATP (µM⁻¹) and oleate (µM) concentrations varied as indicated; B. ATP concentrations were held constant at 10mM and CoA (µM⁻¹) and oleate (µM) concentrations varied as indicated; and C. Oleate concentrations were held constant at 200µM and CoA (µM⁻¹) and ATP (µM) concentrations varied as indicated. V_o⁻¹ values are (nmol/min/mg protein)⁻¹. Shown are representative experiments of an n=4.

Figure 5. Initial velocity studies of the acyl-CoA synthetase reaction in the presence of end products using 1.5µg purified Faa1p

Double reciprocal plots where A. CoA and oleate concentrations were held constant at 20µM and 50µM respectively and the concentrations of ATP (µM⁻¹) and AMP (mM) varied as indicated; B. CoA and ATP concentrations were held constant at 20µM and 5µM respectively and the concentrations of oleate (µM⁻¹) and pyrophosphate (PP_i) (mM) varied as indicated; and C. ATP and oleate concentrations were held constant at 100µM and 50µM respectively and the concentrations of CoA (µM⁻¹) and AMP (mM) varied as indicated. V_o⁻¹ values are (nmol/min/mg protein)⁻¹. Shown are representative experiments of an n=4.