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

Abstract

The properties of the enzyme alkyl dihydroxyacetone phosphate synthase (1-O-acyl dihydroxyacetone 3-phosphate + ROH --> 1-O-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 CHCl3-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 Mg2+ 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 (16:0) dihydroxyacetone-P, with somewhat less activity with longer octadecanoyl (18:0) and much less (40% of 16:0) activity with tetradecanoyl (14:0) dihydroxyacetone-P. Preliminary studies indicate that a Schiffs base intermediate of the enzyme with the keto substrate is probably not formed, since NaBH4 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.