Intermediate channeling on the trifunctional beta-oxidation complex from pig heart mitochondria.

The kinetic properties of the purified trifunctional beta-oxidation complex (TOC) from pig heart mitochondria were analyzed with the aim of elucidating the functional consequence of having three sequentially acting enzymes of beta-oxidation associated in one complex. The kinetic parameters of TOC and of the component enzymes of TOC, long-chain enoyl-CoA hydratase, long-chain 3-hydroxyacyl-CoA dehydrogenase, and long-chain 3-ketoacyl-CoA thiolase, were determined with substrates having acyl chains with 16 carbon atoms. Quantification by high performance liquid chromatography of intermediates formed during the degradation of 2-trans-hexadecanoyl-CoA to myristoyl-CoA and acetyl-CoA by TOC revealed the accumulation of 3-hydroxyhexadecanoyl-CoA, whereas 3-ketohexadecanoyl-CoA was undetectable. The observed rates of NADH and acetyl-CoA formation were higher than the theoretical rates calculated by use of the kinetic parameters and measured concentrations of intermediates. When the sequence of reactions catalyzed by TOC was inhibited by acetyl-CoA, the steady-state concentration of the 3-hydroxyacyl-CoA intermediate was not affected, whereas a small amount of 3-ketohexadecanoyl-CoA was detected. The differences between observed and predicted reaction rates and between measured and expected concentrations of intermediates are best explained by the operation of a channeling mechanism. As a consequence of intermediate channeling between the active sites on the complex, more coenzyme A is available in the mitochondrial matrix and metabolites like 3-ketoacyl-CoA thioesters, which are strong inhibitors of several beta-oxidation enzymes, do not accumulate.

The relationship between mitochondrial activation and toxicity of some substituted carboxylic acids.

The activation of 4-bromocrotonic acid, 4-bromo-2-octenoic acid, valproic acid, and 3-methylglycidic acid by conversion to their CoA thioesters and the effects of these carboxylic acids on palmitoylcarnitine-supported respiration were studied with rat liver and rat heart mitochondria. 4-Bromocrotonic acid was activated by both liver and heart mitochondria, whereas 4-bromo-2-octenoic acid and valproic acid were only activated by liver mitochondria. 3-Methylglycidic acid was not a substrate of mitochondrial activation. All of the carboxylic acids that were activated also inhibited palmitoylcarnitine-supported respiration. 3-Methylglycidoyl-CoA was found to irreversibly inhibit 3-ketoacyl-CoA thiolase in a concentration-dependent and time-dependent manner. Together, these results lead to the conclusion that substituted medium-chain carboxylic acids, which enter mitochondria directly, may inhibit beta-oxidation as long as they are activated and perhaps further metabolized in the mitochondrial matrix to compounds that sequester CoA and/or inhibit beta-oxidation enzymes. Liver is more susceptible to inhibition by such xenobiotic carboxylic acids due to the broader substrate specificity of its mitochondrial medium-chain acyl-CoA synthetase (EC 6.2.1.2).

Specific assay of medium-chain acyl-CoA dehydrogenase based on the spectrophotometric measurement of product formation.

A spectrophotometric method for assaying medium-chain acyl-CoA dehydrogenase is described. The assay measures at 308 nm the formation of cinnamoyl-CoA from 3-phenylpropionyl-CoA in the presence of phenazine methosulfate as electron acceptor. Apparent kinetic constants (Km, Vmax) determined with 3-phenylpropionyl-CoA are similar to constants obtained with octanoyl-CoA, the preferred substrate of this enzyme. The assay is specific for medium-chain acyl-CoA dehydrogenase because long-chain and short-chain acyl-CoA dehydrogenases exhibit little or no activity with 3-phenylpropionyl-CoA as substrate. Since absorbance changes at 308 nm caused by other reactions are less than 5% of the absorbance change due to cinnamoyl-CoA formation catalyzed by medium-chain acyl-CoA dehydrogenase, the assay can be used to measure the activity of this enzyme in crude tissue homogenates. Specific activities of medium-chain acyl-CoA dehydrogenase determined by use of this assay in homogenates of rat liver, heart, and leukocytes were found to be 29, 68, and 2.1 mU/mg of protein, respectively.