Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.3.99.3 (acyl-CoA dehydrogenase)
1,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mechanism of interflavin electron transfer between pig kidney general acyl-CoA dehydrogenase (GAD) and its physiological acceptor, electron-transferring flavoprotein (ETF), has been studied by static and stopped-flow absorbance and fluorescence measurements. At 3 degrees C, pH 7.6, reoxidation of the dehydrogenase (stoichiometrically reduced by octanoyl-CoA) by ETF is multiphasic, consisting of two rapid phases (t1/2 of about 20 and 50 ms), a slower phase half-complete in about 1 s, and a final reaction with a half-time of 20 s. Only the two most rapid phases are significant in turnover. This complicated reaction course was dissected by examining the rates of plausible individual steps, e.g., GAD2e X P + ETF1e, GAD1e X P + ETFox, and GAD1e X P + ETF1e (where P represents the product, octenoyl-CoA, and the subscripts indicate the redox state of the flavin). Rapid reaction and static fluorescence measurements, in all cases, showed that the final equilibrium mixture included appreciable levels of oxidized ETF. This was confirmed by measuring the reverse reactions, e.g., ETF1e + GADox X P, ETF1e + GAD1e X P, and ETF2e + GADox X P. These data support the following overall scheme for the reaction of GAD2e X P with ETFox: The first and second phases correspond to reoxidation of GAD2e X P in two successive one-electron steps requiring two molecules of ETFox. This results in a rapid rise in absorbance at 370 nm where the red anionic radicals of both product-complexed dehydrogenase and ETF absorb strongly.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interflavin oxidation-reduction reactions between pig kidney general acyl-CoA dehydrogenase and electron-transferring flavoprotein. 407 29

Pig kidney general acyl-CoA dehydrogenase is rapidly, stoichiometrically, and irreversibly inactivated by the acetylenic thio ester 2-octynoyl coenzyme A (2-octynoyl-CoA). The inhibitor binds initially to the dehydrogenase with a 10-nm red shift and increased resolution of the flavin chromophore, followed by the generation of a charge-transfer complex between some form of the bound inhibitor and oxidized flavin (lambda max 800 nm; epsilon app = 4.5 mM-1 cm-1; k1 = 1.07 min-1, at pH 7.6, 25 degrees C). The rate of formation of the long wavelength band is increased markedly with increasing pH (pKapp = 7.9). This intermediate then decays with release of about 0.6 mol of CoASH at pH 7.6, yielding a final form with a spectrum typical of bound oxidized flavin. Both irreversible inactivation and covalent modification of the protein occur prior to the decay of the long wavelength species. The modified dehydrogenase is not reduced on prolonged anaerobic incubation with the substrate octanoyl-CoA. The inactive enzyme is unusually resistant to dithionite reduction but may be readily photoreduced via the blue semiquinone to the dihydroflavin form. This reduced enzyme is rapidly reoxidized by electron-transferring flavoprotein, the physiological electron acceptor of the dehydrogenase. General acyl-CoA dehydrogenase is also inactivated by 2-pentynoyl- and 2-pentadecynoyl-CoA with formation of an 800-nm band of lower intensity and by propiolyl-CoA, phenylpropiolyl-CoA, and 2-octynoylpantetheine without the appearance of detectable intermediate species. These data are compared with the behavior of acyl-CoA dehydrogenases toward mechanism-based inactivators carrying an acetylene function at C-3, e.g., 3-butynoyl-CoA.
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PMID:Inactivation of general acyl-CoA dehydrogenase from pig kidney by 2-alkynoyl coenzyme A derivatives: initial aspects. 408 3

Pig kidney general acyl-CoA dehydrogenase is markedly stabilized against loss of flavin and activity in 7.3 M-urea or at 60 degrees C upon reduction with sodium dithionite or octanoyl-CoA. Electron transferring flavoprotein is similarly stabilized, whereas egg white riboflavin-binding protein loses flavin more readily on reduction. These and other data support the anticipated correlation between the kinetic stability of the holoproteins and the oxidation-reduction potential of their bound flavins.
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PMID:The influence of oxidation-reduction state on the kinetic stability of pig kidney general acyl-CoA dehydrogenase and other flavoproteins. 651 65

Kinetic methods for studying the reactions of the "general" fatty acyl CoA dehydrogenase under three sets of substrate and enzyme concentration conditions have been developed. The reaction of butyryl-CoA and electron transfer flavoprotein (ETF) can be studied either under steady-state conditions with enzyme at catalytic concentration or under single-turnover conditions with enzyme in excess. Under the latter conditions, acyl-CoA dehydrogenase acts both as a catalyst and an ultimate electron-transfer acceptor. The reductive half-reaction of butyryl-CoA and enzyme can also be studied in a separate kinetic experiment. Comparison of the pH dependences of the rate constants and isotope effects of the steady-state reaction of butyryl-CoA and ETF with the same parameters for the reductive half-reaction is consistent with a mechanism involving transfer of electrons from butyryl-CoA to ETF within a ternary complex. An alternative mechanism in which the reductive half-reaction takes place prior to the binding and reaction of ETF seems unlikely because the pH 8.5 isotope effect on the reductive half-reaction is much larger than that on the complete reaction in spite of the fact that the rates of the reactions are comparable. The pH dependence of the Km for substrate and KI for inhibitor is consistent with a mechanism for transfer of electrons within the ternary complex which involves protonation of the C = O group of substrates. The protonation labilizes the C-2 proton and base catalysis of the removal of the C-2 proton results in the production of the active enzyme-substrate species, namely the C-2 anion of substrate.
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PMID:Kinetic methods for the study of the enzyme systems of beta-oxidation. 663 77

Three children in two families presented in early childhood with episodes of illness associated with fasting which resembled Reye's syndrome: coma, hypoglycemia, hyperammonemia, and fatty liver. One child died with cerebral edema during an episode. Clinical studies revealed an absence of ketosis on fasting (plasma beta-hydroxybutyrate less than 0.4 mmole/liter) despite elevated levels of free fatty acids (2.6-4.2 mmole/liter) which suggested that hepatic fatty acid oxidation was impaired. Urinary dicarboxylic acids were elevated during illness or fasting. Total carnitine levels were low in plasma (18-25 mumole/liter), liver (200-500 nmole/g), and muscle (500-800 nmole/g); however, treatment with L-carnitine failed to correct the defect in ketogenesis. Studies on ketone production from fatty acid substrates by liver tissue in vitro showed normal rates from short-chain fatty acids, but very low rates from all medium and long-chain fatty acid substrates. These results suggested that the defect was in the mid-portion of the intramitochondrial beta-oxidation pathway at the medium-chain acyl-CoA dehydrogenase step. A new assay for the electron transfer flavoprotein-linked acyl-CoA dehydrogenases was used to test this hypothesis. This assay follows the decrease in electron transfer flavoprotein fluorescence as it is reduced by acyl-CoA-acyl-CoA dehydrogenase complex. Results with octanoyl-CoA as substrate indicated that patients had less than 2.5% normal activity of medium-chain acyl-CoA dehydrogenase. The activities of short-chain and isovaleryl acyl-CoA dehydrogenases were normal; the activity of long-chain acyl-CoA dehydrogenase was one-third normal. These results define a previously unrecognized inherited metabolic disorder of fatty acid oxidation due to deficiency of medium-chain acyl-CoA dehydrogenase.
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PMID:Medium-chain acyl-CoA dehydrogenase deficiency in children with non-ketotic hypoglycemia and low carnitine levels. 664 97

Rats treated with six to eight doses (80 mg/kg, i.p.) of 4-pentenoic acid, an inhibitor of mitochondrial fatty acid oxidation in vitro, during a 48-hr starvation period developed microvesicular fatty infiltration of the liver similar to that observed in Reye's Syndrome. Hepatic triglycerides were elevated an average of 5-fold, although considerable variability was found between individual rats. Fed rats did not develop fatty liver upon similar treatment with pentenoic acid. Liver mitochondria isolated from rats with pentenoic acid-induced fatty liver showed a persistent inhibition of fatty acid oxidation. Rates of oxidation of palmitoylcarnitine and decanoylcarnitine were decreased about 70%, while that of octanoylcarnitine was decreased 50%. Carnitine-independent oxidation of octanoate was also inhibited. Oxidation rates for substrates other than fatty acids, including glutamate, succinate, pyruvate, and alpha-ketoglutarate, were unaffected. Measurements of flavoprotein reduction in intact mitochondria indicated that neither palmitoylcarnitine nor palmitoyl CoA plus L-carnitine could elicit reduction of acyl-CoA dehydrogenase and electron transferring flavoprotein in mitochondria from rats with pentenoic acid-induced fatty liver. These results support a site of inhibition of mitochondrial beta-oxidation at the level of acyl-CoA dehydrogenase for pentenoic acid treatment in vivo, and they suggest a role for nutritional or hormonal factors in the metabolic disposition of pentenoic acid in vivo and in the development of fatty liver.
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PMID:Inhibition of mitochondrial fatty acid oxidation in pentenoic acid-induced fatty liver. A possible model for Reye's syndrome. 671 30

The effects of pH and ionic strength on the steady state kinetic parameters for reduction of electron transfer flavoprotein (ETF) by general acyl-CoA dehydrogenase were determined. The effect of pH on the turnover number (TN) of the reaction indicates the participation of an essential base with a pK alpha of 6.9. The KmETF of the dehydrogenase is invariant between pH 5.4 and 8.5, but increases 40-fold between pH 8.5 and 9.8. The parameter TN/KmETF follows the limiting Bronsted equation (In TN/KmETF = ln ko + 2.34ZAZB I 1/2) at ionic strength values between 0.01 and 0.125 M, indicating complementary charge interactions between the two flavoproteins. Covalent modifications of amino groups of ETF with trinitrobenzene sulfonate and acetic anhydride remove positive charges and result in an increase in KmETF of the dehydrogenase with no change of TN. However, exhaustive acetimidation of ETF amino groups, which maintains cationic charge at modified loci, does not alter the steady state kinetic parameters of the reaction. These results, in conjunction with previous chemical covalent modifications of dehydrogenase carboxyl residues (Frerman, F. E., Mielke, D., and Huhta, K. (1980) J. Biol. Chem. 255, 2199-2202), indicate that general acyl-CoA dehydrogenase and ETF interact in an electrostatic manner.
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PMID:The effects of pH, ionic strength, and chemical modifications on the reaction of electron transfer flavoprotein with an acyl coenzyme A dehydrogenase. 686 54

2-Methyl-branched chain acyl-CoA dehydrogenase was purified to homogeneity from rat liver mitochondria. The native molecular weight of the enzyme was estimated to be 170,000 by gel filtration. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis both with and without 2-mercaptoethanol, the enzyme showed a single protein band with Mr = 41,500, suggesting that this enzyme is composed of four subunits of equal size. Its isoelectric point was 5.50 +/- 0.2, and A1%280 nm was 12.5. This enzyme contained protein-bound FAD. The purified enzyme dehydrogenated S-2-methylbutyryl-CoA and isobutyryl-CoA with equal activity. The activities with each of these compounds were co-purified throughout the entire purification procedure. This enzyme also dehydrogenated R-2-methylbutyryl-CoA, but the specific activity was considerably lower (22%) than that for the S-enantiomer. The enzyme did not dehydrogenate other acyl-CoAs, including isovaleryl-CoA, propionyl-CoA, butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA, at any significant rate. Apparent Km and Vmax values for S-2-methylbutyryl-CoA were 20 microM and 2.2 mumol min-1 mg-1, respectively, while those for isobutyryl-CoA were 89 microM and 2.0 mumol min-1 mg-1 using phenazine methosulfate as an artificial electron acceptor. The enzyme was also active with electron transfer flavoprotein. Tiglyl-CoA and methacrylyl-CoA were identified as the reaction products from S-2-methylbutyryl-CoA and isobutyryl-CoA, respectively. 2-Ethylacrylyl-CoA was produced from R-2-methylbutyryl-CoA. Tiglyl-CoA competitively inhibited the activity with both S-2-methylbutyryl-CoA and isobutyryl-CoA with a similar Ki. The enzyme activity was also severely inhibited by several organic sulfhydryl reagents such as N-ethylmaleimide, p-hydroxymercuribenzoate, and methyl mercury iodide. The pattern and degree of inhibition were essentially identical for both substrates. The purified 2-methyl-branched chain acyl-CoA dehydrogenase was immunologically distinct from isovaleryl-CoA-, short chain acyl-CoA-, medium chain acyl-CoA-, or long chain acyl-CoA dehydrogenase.
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PMID:Purification and characterization of 2-methyl-branched chain acyl coenzyme A dehydrogenase, an enzyme involved in the isoleucine and valine metabolism, from rat liver mitochondria. 687 97

1. A reflection spectrometric method was developed which allowed the simultaneous measurement of flavoprotein absorption and fluorescence on an in vitro preparation of brown adipose tissue. 2. From their spectral characteristics and from the effects of substrates and a metabolic inhibitor (amytal) it was shown that the absorption and fluorescence signals are associated with different flavoproteins. 3. The fluorescence signal is mainly due to changes in the redox state of NADH dehydrogenase, and the absorption signal to changes in redox state of he flavoproteins in the acyl-CoA dehydrogenase pathway. 4. The results suggest that changes in the flavoprotein redox state in response to electrical nerve stimulation, exogenous norepinephrine and substrate addition reflect changes in the metabolic activity of the tissue. These responses were studied in the postnatal period. 5. The amplitude of the tissue response to either nerve stimulation or norepinephrine administration is already maximal at birth and decreases in animals 50 days old. The frequency of nerve stimulation of the concentration of norepinephrine required to produce a half maximum response is significantly higher for the new-born as compared to 13 day and 50 day old animals. 6. For small stimulation intensities a steady state oxidation of the NADH dehydrogenase concomitant with a steady state reduction of the flavoproteins in the acyl-CoA dehydrogenase pathway was recorded. 7. It is concluded that in rats less than 12 hours old, brown adipose tissue is functionally innervated although previous histochemical studies had failed to detect nerve terminals containing catecholamines at this early age.
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PMID:Postnatal development of sympathetic innervation of rat brown adipose tissue reevaluated with a method allowing for monitoring flavoprotein redox state. 713 28

Weanling rats were fed a riboflavin-deficient diet. The mitochondrial fatty acid oxidation in liver was depressed in riboflavin deficiency but restored after supplementation of riboflavin. Among the enzymes involved in this system, only the acyl-CoA dehydrogenase (EC 1.3.99.2 and 1.3.99.3) activities varied with the change in fatty acid oxidation. An accumulation of the apoforms of acyl-CoA dehydrogenases was found in riboflavin deficiency. The levels of electron transfer flavoprotein and other enzymes involved in the beta-oxidation system remained unchanged. The peroxisomal fatty acid oxidation and levels of individual enzymes of this system remained constant. No accumulation of the apoform of acyl-CoA oxidase was observed under simple, riboflavin-deficient conditions. However, accumulation of a large amount of apo-acyl-CoA oxidase was observed when the peroxisomal system was induced by administration of a peroxisome proliferator, di(2-ethylhexyl)phthalate, under riboflavin-deficient conditions.
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PMID:Riboflavin deficiency and beta-oxidation systems in rat liver. 714 48


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