Thermodynamic bases for fatty acid ethyl ester synthase: catalyzed esterification of free fatty acid with ethanol and accumulation of fatty acid ethyl esters

Abstract

Myocardial [rabbit] homogenates rapidly synthesize fatty acyl ethyl esters from nonesterified fatty acid and ethanol in the absence of CoA or ATP, and the enzyme catalyzing this reaction, fatty acid ethyl ester synthase, was purified 5400-fold to homogeneity (preceding paper in this issue). To define the factors permitting this de novo synthesis of ester bonds and the consequent accumulation of fatty acyl ethyl esters in myocardium, thermodynamic parameters relevant to the kinetics and equilibria of this reaction were determined and the rates of synthesis of ethyl oleate, in both the presence and absence of purified enzyme catalyst, and the physical properties of the product, ethyl oleate, were specifically characterized in an aqueous milieu. Compared to the reaction of ethanol and oleate in the absence of catalyst, fatty acid ethyl ester synthase enhanced the rate of ethyl oleate synthesis by reducing the free energy of activation (.DELTA.G*) from 32.5 to 19.9 kcal/mol, effected in large part by a positive entropy shift, .DELTA.S*enz - .DELTA.S*uncat = 23.9 cal/(mol .cntdot. deg). Rate constants in the presence and absence of enzyme at 37.degree. C were 6 .times. 10-2 s-1 and 7.8 .times. 10-11 M-1 s-1, respectively, indicating a catalytic power of at least 108 M for this enzyme. Kinetic data indicated an enzymatic Vmax of 1.25 nmol/(mg .cntdot. s) (37.degree. C). The equilibrium constant was calculated for the reaction oleate + ethanol .dblarw. ethyl oleate and was 0.095 M-1 at 37.degree. C. Since this equilibrium is slightly thermodynamically unfavorable (.DELTA.G = + 1.45 kcal/mol) even though ethyl esters are known to accumulate in myocardium, the possibility was considered that self-association of the lipid product, ethyl oleate, in an aqueous medium, is a thermodynamically more favorable step permitting net product accumulation. Indeed, aggregation of ethyl oleate occurred in water in a temperature-dependent manner between 20 and 44.degree. C, e.g., at and above 0.7 .mu.M at 37.degree. C, associated with a .DELTA.G.degree. of -11.1 kcal/mol. The synthesis of fatty acyl ethyl esters is rapidly facilitated by fatty acyl ethyl ester synthase, an augmentation of reaction rate that reflects a reduction in .DELTA.G* largely due to a positive entropy change, and accumulation of product, fatty acyl ethyl ester, is a consequence of the thermodynamically favorable self-association of monomeric molecules into a hydrocarbon phase. These results elucidate an entropic rather than enthalpic basis for covalent bond formation in a biologic system that may bear importantly on the biosynthesis of other naturally occurring esters.