Control of mitochondrial beta-oxidation: sensitivity of the trifunctional protein to [NAD(+)]/[NADH] and [acetyl-CoA]/[CoA]

Isolated human mitochondrial trifunctional protein was incubated with 2-hex adecenoyl-CoA, CoA and NAD(+) and the resultant CoA esters measured. Steady state with respect to the concentrations of the intermediates 3-hydroxyhex adecanoyl-CoA and 3-ketohexadecanoyl-CoA and the rate of formation of the p roduct tetradecanoyl-CoA was reached within 4 min. Flux was greatly enhance d by the addition of Tween 20 (0.2% v/v) which stimulated 3-ketoacyl-CoA th iolase activity by over 7-fold. When 3-ketoacyl-CoA thiolase was not stimul ated, 3-hydroxyhexadecanoyl-CoA was the prominent CoA ester accumulated, pr esumably due to inhibition of 3-hydroxyacyl-CoA dehydrogenase activity by a ccumulated 3-ketoacyl-CoA, analogous to the inhibition of short-chain 3-hyd roxyacyl-CoA dehydrogenase by 3-ketoacyl-CoA. When [NAD(+)]/[NADH] was vari ed at a fixed total [NAD(+)+NADH], the overall flux was only inhibited by [ NAD(+)]/[NADH] less than 1. In contrast, when [acetyl-CoA]/[CoA] was varied at a fixed total [CoA], much greater sensitivity was observed. (C) 1998 El sevier Science B.V. All rights reserved.