Dehydration of 3-hydroxyacyl-CoA in brain very-long-chain fatty acid synthesis

Rat brain microsomes actively dehydrate 3-hydroxyacyl-CoAs. Using chemicall y synthesized [1-C-14] (R,S) 3-hydroxyeicosanoyl-CoA, we investigated the b iochemical characteristics of the dehydration and reduction steps of stearo yl-CoA elongation. The reaction products, separated and identified as trans2,3-enoyl-CoAs and, in the presence of NADPH, as saturated acylCoAs, were released from the en zyme as thioesters which were partly hydrolysed. A kinetic analysis of the two coupled reactions showed that the 3-hydroxyacyl-CoA dehydrase catalysed a reversible reaction with kinetic constants of about 0.045 min(-1) for fo rward reaction (dehydration) and 0.025 min(-1) for reverse reaction (hydrat ion); V-max of the dehydration reached 20 nmoles/min/mg and the apparent Km was 44 mu M. In the presence of NADPH, the kinetic constants for the dehyd rase were unchanged and that for the trans2,3-enoyl-CoA reductase was 0.025 min(-1). The relative proportion of trans2,3-enoyl-CoA and saturated acyl- CoA depended on the protein amount. An inhibition of the reduction step was observed for substrate concentrations above 15 mu M. The 3-hydroxyacyl-CoA dehydrase used (R) rather than (S) 3-hydroxyacyl-CoA. Furthermore, the elongation of (R) 3-hydroxyeicosanoyl-CoA yielded saturat ed very-long-chain acyl-CoA. These results demonstrated that 3-hydroxyacyl- CoAs entered the elongating complex exclusively at the level of the dehydra se and not of the condensing enzyme. (C) 1999 Elsevier Science Ltd. All rig hts reserved.