Dehydration of 3-hydroxyacyl-CoA in brain very-long-chain fatty acid synthesis.
Rat brain microsomes actively dehydrate 3-hydroxyacyl-CoAs. Using chemically synthesized [1-(14)C] (R,S) 3-hydroxyeicosanoyl-CoA, we investigated the biochemical characteristics of the dehydration and reduction steps of stearoyl-CoA elongation. The reaction products, separated and identified as trans2,3-enoyl-CoAs and, in the presence of NADPH, as saturated acyl-CoAs, were released from the enzyme 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 forward reaction (dehydration) and 0.025 min(-1) for reverse reaction (hydration); Vmax of the dehydration reached 20 nmoles/min/mg and the apparent Km was 44 microM. In the presence of NADPH, the kinetic constants for the dehydrase 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 microM. The 3-hydroxyacyl-CoA dehydrase used (R) rather than (S) 3-hydroxyacyl-CoA. Furthermore, the elongation of (R) 3-hydroxyeicosanoyl-CoA yielded saturated very-long-chain acyl-CoA. These results demonstrated that 3-hydroxyacyl-CoAs entered the elongating complex exclusively at the level of the dehydrase and not of the condensing enzyme.
Knoll
A
A
CNRS-UMR 5544, Université Victor Segalen Bordeaux 2, France. anja.knoll@biomemb.u-bordeaux2.fr
Bessoule
J J
JJ
Sargueil
F
F
Cassagne
C
C
D044944:Acyl-CoA Dehydrogenases; D000818:Animals; D001921:Brain / Q000201:enzymology*; D003065:Coenzyme A / Q000737:chemistry / Q000378:metabolism; D004952:Esters; D044943:Fatty Acid Desaturases / Q000378:metabolism; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D006836:Hydro-Lyases / Q000378:metabolism*; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017550:Spectroscopy, Fourier Transform Infrared; D013379:Substrate Specificity; D014867:Water / Q000378:metabolism
D004952:Esters; D005227:Fatty Acids; D014867:Water; D009249:NADP; D044943:Fatty Acid Desaturases; D044944:Acyl-CoA Dehydrogenases; C100154:acyl-CoA dehydrogenase (NADP+); C065185:D-3-hydroxyacyl CoA dehydratase; D006836:Hydro-Lyases; D003065:Coenzyme A