Assay and properties of the enzyme catalyzing the biosynthesis of 1-O-alkyl dihydroxyacetone 3-phosphate.

The properties of the enzyme alkyl dihydroxyacetone phosphate synthase (1-O-acyl dihydroxyacetone 3-phosphate + ROH 1-0-alkyl dihydroxyacetone 3-phosphate + fatty acid) in Ehrlich ascites tumor cell microsomes were studied utilizing a new assay procedure. The assay is based on the difference of partition coefficient between alkyl dihydroxyacetone-P and hexadecanol in an alkaline two-phase CHC&-methanol-water system. The product of the enzymatic reaction was characterized by different methods as alkyl dihydroxyacetone-P. At optimum concentration of the substrates (acyl dihydroxyacetone-P and hexadecanol), detergents or M%f and ATP do not stimulate the reaction. The kinetic properties of the reaction were found to be complex due to the stimulation of enzyme activity at low acyl dihydroxyacetone-P concentration but inhibition at the high concentration of the same substrate. The enzyme accepts a variety of long-chain primary alcohols as substrate without much specificity. With different acyl dihydroxyacetone-P, the highest activity was obtained with hexadecanoyl(16:O) dihydroxyacetoneP, with somewhat less activity with longer octadecanoyl (180) and much less (40% of 16:0) activity with tetradecanoyl(14:O) dihydroxyacetone-P. Preliminary studies indicate that a Schiff’s base intermediate of the enzyme with the keto substrate is probably not formed, since NaBH, inhibited the enzyme to the same extent in the presence or absence of acyl dihydroxyacetone-P. Fatty acids inhibit the reaction and an exchange of fatty acid with acyl dihydroxyacetone-P in the presence of the enzyme preparation was observed. Heat-inactivation studies indicate that alkyl dihydroxyacetone-P synthase is probably also catalyzing this fatty acid exchange reaction. Based on these findings, an enzyme-bound dihydroxyacetone-P intermediate is proposed.