Mechanism of ether bond formation by alkyldihydroxyacetone-P synthase

Alkyldihydroxyacetone-P (alkyl-DHAP) synthase catalyzes a unique reaction in which the fatty acid ester of acyl-DHAP is cleaved and replaced by a fatty alcohol to form alkyl-DHAP, the first ether-linked intermediate in the biosynthesis of the alkyl- or alk-l-enylglycerolipids. In order to study this reaction mechanism in greater detail, this enzyme was solubilized for the first time from Ehrlich ascites cell microsomal membranes using Triton X-100 and acetone, and purified 1000-fold by chromatography on DEAE-cellulose, QAE-Sephadex, Matrex Red, and hydroxylapatite with the aid of a new rapid assay using DEAE paper disks.

The acyl exchange activity in which [14_C] palmitic acid is incorporated into acyl-DHAP, and an analogous alkyl exchange reaction, in which [14_C] hexadecanol is incorporated into alkyl-DHAP, copurified with the forward reaction strongly supporting a ping-pong mechanism for alkyl-DHAP synthase. Further evidence was obtained by showing that palmitic acid is a competitive inhibitor, with respect to hexadecanol, of the forward reaction.

Highly purified alkyl-DHAP synthase slowly exchanged ³H from acyl-[1-R- ³H] DHAP in the absence of cosubstrate. Addition of hexadecanol increased the ³H exchange rate by an amount equal to the rate of alkyl-DHAP formation. Similarly, the increase in rate of ³H exchange by addition of palmitic acid matched the rate of the acyl exchange reaction.

We have proposed that upon binding of acyl-DHAP to alkyl-DHAP synthase, the pro-R hydrogen at carbon 1 is exchanged by an enolization of the ketone, followed by release of the acyl moiety to form an activated enzyme-DHAP intermediate. Carbon-1 of the intermediate is believed to carry a positive charge which may be stabilized by an essential sulfhydryl group; the incoming alkoxide ion reacts at carbon-1 to form alkyl-DHAP.