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-hexadecenoyl-CoA, CoA and NAD+ and the resultant CoA esters measured. Steady state with respect to the concentrations of the intermediates 3-hydroxyhexadecanoyl-CoA and 3-ketohexadecanoyl-CoA and the rate of formation of the product tetradecanoyl-CoA was reached within 4 min. Flux was greatly enhanced by the addition of Tween 20 (0.2% v/v) which stimulated 3-ketoacyl-CoA thiolase activity by over 7-fold. When 3-ketoacyl-CoA thiolase was not stimulated, 3-hydroxyhexadecanoyl-CoA was the prominent CoA ester accumulated, presumably due to inhibition of 3-hydroxyacyl-CoA dehydrogenase activity by accumulated 3-ketoacyl-CoA, analogous to the inhibition of short-chain 3-hydroxyacyl-CoA dehydrogenase by 3-ketoacyl-CoA. When [NAD+]/[NADH] was varied 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.