Formation of tritiated O-alkyl lipid from acyldihydroxyacetone phosphate in the presence of tritiated water

Abstract

Previous studies from this laboratory on the mechanism of O-alkyl bond formation using a microsomal system from Tetrahymena pyriformis have shown that O-alkyl lipid synthesized from dihydroxyacetone phosphate has exchanged one hydrogen stereospecifically from the 1-sn position of the glycerol moiety. Indirect evidence suggested that acyldihydroxyacetone phosphate, an intermediate in )-alkyl lipid synthesis, is probably not the locus of the exchange. In the present study in was shown that stable acyldihydroxyacetone phosphate incubated in the presence of tritiated water and Tetrahymena microsomes does not become tritiated. When hexadecanol is added to the system O-alkyl lipid is produced which has incorporated one atom of hydrogen for each mole of hexadecanol at all time periods examined. Experiments in Ehrlich ascites tumor cells have shown that the hydrogen exchange also occurs in a mammalian system. The results indicate that the mechanism of O-alkyl lipid ether bond formation involves a hydrogen exchange and that this exchange occurs after the formation of acyldihydroxyacetone phosphate.

Use of [1 or 3-3H, U-14c]glucose to estimate the synthesis of glycerolipids via acyl dihydroxyacetone phosphate

“…1975 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS of ether-containing glycerolipids thus supporting the view that they are synthesized via acyl DHAP (3). In subcellular fractions, this C-I hydrogen has been found to exchange with water during conversion of acyl DHAP to alkyi DHAP(21) and the data here support the occurrence of this exchange in intact cells.A similar exchange of the C-I hydrogen can occur during the DHAP to glyceraldehyde-3-phosphate equilibrium catalyzed by triose phosphate isomerase (EC 5.3.1.1). The appearance of 3H in C-I of GP and saponifiable glycerolipids indicates this latter exchange is incomplete, as has been previously reported for adipose tissue(6).…”

Glyceryl-ether monooxygenase [EC 1.14.16.5]. A microsomal enzyme of ether lipid metabolism

“…This reaction is unique and complex because it catalyzes several steps. It was studied extensively by Snyder, 159-165 Friedberg 156,[166][167][168][169][170] and Hajra, [171][172][173][174][175] and their coworkers. The important features of the reaction were reported many times previously 165 and briefly are: (i) the acyloxy group in acyl-DHAP with its two oxygen atoms is completely displaced by the alkoxy group of the incoming fatty alcohol as shown by retention of the labeled oxygen atom when […”

“…18 O]-hexadecanol was used, 160 (ii) the 1-pro-R hydrogen atom of acyl-DHAP is exchanged with water of the solvent 162,166,167 with retention of configuration at the carbon atom bearing the acyloxy group (at C-1), 168,172 (iii) the enzyme binds covalently to acyl-DHAP. 162,164,165 Friedberg et al 169 suggested an intermediate with both acyloxy and alkoxy groups attached to DHAP whereas Davis and Hajra 171 preferred a Ping-Pong Mechanism where an activated enzyme-DHAP complex is formed with release of the fatty acid which is then attacked by the alcohol followed by detachment from the enzyme.…”

Noncanonical Reactions of Flavoenzymes

“…Early mechanistic work was done with microsomal fractions and solubilized enzyme [91,92]. It was shown by tritium exchange experiments that the pro - R -H at the C1 position was abstracted and exchanged with the solvent [93–95]. Additionally, it was demonstrated by isotope labeling experiments that both of the oxygens on the fatty acid are removed during the acyl cleavage and that the oxygen in the ether bond is donated by the alcohol substrate [96,97].…”