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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)

Aspects of the binding and dehydrogenation of acyl-CoA thiol esters by the general acyl-CoA dehydrogenase from pig liver were investigated using a dead-end inhibitor, S-octyl-CoA, several alternate substrates, and three active site-directed inhibitors. Experiments with S-octyl-CoA indicate that the carbonyl group of acyl-CoA thiol esters is not absolutely required for binding to the enzyme. However, the mode of binding of the 8-carbon thiol ether can be distinguished from the mode of binding of the enoyl-CoA product, octenoyl-CoA. Octanoyl pantetheine, octanoyl-etheno-CoA, and octanoyl-3'-dephospho-CoA are alternate substrates of the dehydrogenase. Steady state kinetic constants obtained with these alternate substrates indicate that the adenosine 5'-diphosphate, but not the 3'-phosphate, of the nucleotide moiety of acyl-CoA substrates contribute to the tight binding of the substrates. The substrate analogs 3'-butynoyl-CoA and 3-octynoyl-CoA are active site-directed, mechanism-based irreversible inhibitors of the dehydrogenase. These inhibitors covalently modify the apoprotein rather than the flavin. This finding and the fact that 2,3-octadienoyl-CoA also completely and irreversibly inhibits the enzyme indicate that th 3-acetylenic thiol esters inhibit the enzyme by a mechanism involving: (1) base-catalyzed abstraction of a protein at C-2 followed by isomerization to the allene carbanion, (2) protonation of the carbanion, and (3) attack of a nucleophile in the enzyme-active site on C-3 of the 2,3-dienoyl-CoA. The data show that the alkynoyl-CoA's are activated and bound at the active site of the enzyme. The results suggest that abstraction of a proton at C-2 of acyl-CoA substrates is the initial step in the catalytic pathway of dehydrogenation of substrates by the enzyme.
J Biol Chem 1980 Dec 10
PMID:Enzyme-activated inhibitors, alternate substrates, and a dead end inhibitor of the general acyl-CoA dehydrogenase. 744 May 36

Utilization of fatty acids for energy varies among mammalian tissues and during development due to changes in expression of enzymes of mitochondrial beta oxidation. To discern whether two related nuclear genes are expressed similarly, the tissue distribution and developmental profile of the rat long- and medium-chain acyl-CoA dehydrogenase (LCAD and MCAD) mRNAs were compared. A 1451 base full-length LCAD cDNA from neonatal rat aorta was used to study mRNA accumulation in adult and fetal rat tissues. LCAD and MCAD mRNAs were expressed in aorta, heart, and brown fat at levels 8-40 fold greater than in liver, kidney, and duodenum. Brain, placenta, ovary, testes, and skeletal muscle showed the least mRNA. Western blots of adult tissues with anti-rat LCAD antiserum showed corresponding amounts of LCAD protein subunits. LCAD mRNA was detectable in heart, liver, kidney, and brain of fetal rats and increased with age. LCAD and MCAD mRNAs were present in brown fat in 2-10 fold greater amounts compared to other tissues from the newborn period to the end of the weaning period. The high level of expression of LCAD and MCAD mRNA in aorta, heart, and brown fat likely reflects the high energy requirements of those tissues. Differential expression of LCAD and MCAD mRNAs reflects not only inherent gene prescribed programs, but also external influences such as hormones and diet.
Biochim Biophys Acta 1993 Dec 14
PMID:Tissue specific and developmental expression of rat long-and medium-chain acyl-CoA dehydrogenases. 826 28

The activity of the enzyme acyl-CoA oxidase (EC 1.3.99.3) is influenced by detergents. At concentrations above the critical micellar concentration, Triton X-100, Triton X-114 and Thesit stimulate oxidase activity. Lower concentrations of Triton X-100 and Triton X-114 render the acyl-CoA oxidase less sensitive towards substrate inhibition by palmitoyl-CoA or dec-4-cis-enoyl-CoA. Other detergents inhibited the enzyme activity. CoA was found to be a relatively powerful competitive inhibitor of the enzyme, with a Ki,slope value of 63 +/- 3 microM. This inhibition is dependent on an intact CoA molecule, as dephospho-CoA, dethio-CoA and acetyl-CoA are less potent inhibitors of the enzyme. Dec-2-trans-enoyl-CoA is a product-inhibitor of acyl-CoA oxidase, with a Ki,slope value of 7 +/- 1 microM.
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PMID:Factors which affect the activity of purified rat liver acyl-CoA oxidase. 843 1

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is a major enzyme catalyzing long-chain fatty acids in the first step of mitochondrial beta-oxidation system. Inborn error of this enzyme can cause sudden infant death syndrome and hypertrophic cardiomyopathy is present at a significantly high frequency. To investigate VLCAD deficiency at the genomic DNA level, we cloned the VLCAD gene and analyzed the structure. The gene is about 5.4 kb long and contains 20 exons. We performed mutation analysis in two patients, both having a 105 bp deletion encompassing bases 1078-1182 in cDNA. A point mutation (GT-->AT) at 5' splice site of intron 11 was identified in both patients. This mutation seems to cause skipping of exon 11 corresponding to the 105 bp deletion. This is the first documentation of aberrant splicing in the VLCAD gene.
Biochem Biophys Res Commun 1995 Dec 26
PMID:Genomic DNA organization of human mitochondrial very-long-chain acyl-CoA dehydrogenase and mutation analysis. 855 25

The intense charge transfer complex between the enolate of 3-thia-octanoyl-CoA and the oxidized flavin of the medium-chain acyl-CoA dehydrogenase is discharged by the ferricenium ion with irreversible inactivation of the enzyme. Charge transfer complex formation is a necessary, but insufficient, condition for oxidative inactivation: the 3-oxa-octanoyl-CoA complex is also inactivated, whereas the comparable trans-3-octenoyl-CoA species is not. Complete inactivation of the dehydrogenase with 3-thia-octanoyl-CoA requires 1 molecule of thioester and apparently 3 molecules of ferricenium hexafluorophosphate. Experiments with 8-Cl-FAD substituted enzyme and the crystal structure of enzyme.ligand complexes argue that ferricenium ion-mediated oxidation proceeds through the flavin prosthetic group. Synthesis of [2-14C]-3-thia-octanoyl-CoA, followed by isolation of radiolabeled peptide from the modified medium-chain dehydrogenase, showed that inactivation results in labeling the catalytic base, GLU376. Oxidative modification is accompanied by the release of CoASH. A mechanism for inactivation is proposed involving generation of a sulfonium salt which efficiently captures the carboxylate nucleophile.
Biochemistry 1995 Dec 19
PMID:Oxidative inactivation of a charge transfer complex in the medium-chain acyl-CoA dehydrogenase. 884 70

The activities of enzymes in fatty acid oxidation and synthesis in the liver of rats fed soybean phospholipids and soybean oil corresponding to the dietary levels of 3% fatty acid added to the diets containing a saturated fat (coconut oil) and a polyunsaturated fat (safflower oil) at the amounts corresponding to 12% fatty acid levels were compared. Soybean phospholipid compared with soybean oil added to both coconut and safflower oil diets significantly reduced the activities of enzymes in fatty acid synthesis (fatty and synthetase, glucose-6-phosphate dehydrogenase and malic enzyme). However, there were no significant differences in the activities of enzymes in fatty acid oxidation (carnitine palmitoyltransferase, acyl-CoA dehydrogenase and acyl-CoA oxidase) between the groups of rats fed soybean phospholipid and soybean oil added to coconut and safflower oil diets except for one occasion. Soybean phospholipid compared with soybean oil added to coconut oil diet significantly decreased the concentrations of triacylglycerol, cholesterol and phospholipid in the serum and of triacylglycerol and cholesterol in the liver. However, the dietary phospholipid added to safflower oil diet failed to alter these values. These results suggested that the alteration in the rate of fatty acid synthesis, but not oxidation, in the liver is responsible for the lipid-lowering effect of dietary soybean phospholipid added to a saturated fat diet.
J Nutr Sci Vitaminol (Tokyo) 1995 Dec
PMID:Effect of dietary soybean phospholipid and fats differing in the degree of unsaturation on fatty acid synthesis and oxidation in rat liver. 892 36

The acyl-CoA dehydrogenases are a family of flavoenzymes with similar structure and function involved in the metabolism of fatty acids and branched chain amino acids. The degree of overlap in substrate specificity is narrow among these enzymes. The position of the catalytic glutamate, identified as Glu376 in porcine medium chain acyl-CoA dehydrogenase (MCAD), Glu254 in human isovaleryl-CoA dehydrogenase (IVD), and Glu261 in human long chain acyl-CoA dehydrogenase (LCAD), has been suggested to affect substrate chain length specificity. In this study, in vitro site-directed mutagenesis was used to investigate the effect of changing the position of the catalytic carboxylate on substrate specificity in short chain acyl-CoA dehydrogenase (SCAD). Glu368, the hypothetical active site catalytic residue of rat SCAD, was replaced with Asp, Gly, Gln, Arg, and Lys and the wild type and mutant SCADs were produced in Escherichia coli and purified. The recombinant wild type SCAD kcat/K(m) values for butyryl-hexanoyl-, and octanoyl-CoA were 220, 22, and 3.2 microM-1 min-1, respectively, while the Glu368Asp mutant gave kcat/K(m) of 81, 12, and 1.4 microM-1 min-1, respectively, for the same substrates. None of the other mutants exhibited enzyme activity. A Glu368Gly/Gly247Glu double mutant enzyme, which places the catalytic residue at a position homologous to that of LCAD, was also synthesized and purified. It showed kcat/K(m) of 9.3, 2.8, and 1.5 microM-1 min-1 with butyryl-, hexanoyl-, and octanoyl-CoA used as substrates, respectively. These results confirm the identity of Glu368 as the catalytic residue of rat SCAD and suggest that alteration of the position of the catalytic carboxylate can modify substrate specificity.
Biochemistry 1996 Dec 03
PMID:Functional role of the active site glutamate-368 in rat short chain acyl-CoA dehydrogenase. 895 87

Mammalian electron transfer flavoproteins (ETF) are heterodimers containing a single equivalent of flavin adenine dinucleotide (FAD). They function as electron shuttles between primary flavoprotein dehydrogenases involved in mitochondrial fatty acid and amino acid catabolism and the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase. The structure of human ETF solved to 2.1-A resolution reveals that the ETF molecule is comprised of three distinct domains: two domains are contributed by the alpha subunit and the third domain is made up entirely by the beta subunit. The N-terminal portion of the alpha subunit and the majority of the beta subunit have identical polypeptide folds, in the absence of any sequence homology. FAD lies in a cleft between the two subunits, with most of the FAD molecule residing in the C-terminal portion of the alpha subunit. Alignment of all the known sequences for the ETF alpha subunits together with the putative FixB gene product shows that the residues directly involved in FAD binding are conserved. A hydrogen bond is formed between the N5 of the FAD isoalloxazine ring and the hydroxyl side chain of alpha T266, suggesting why the pathogenic mutation, alpha T266M, affects ETF activity in patients with glutaric acidemia type II. Hydrogen bonds between the 4'-hydroxyl of the ribityl chain of FAD and N1 of the isoalloxazine ring, and between alpha H286 and the C2-carbonyl oxygen of the isoalloxazine ring, may play a role in the stabilization of the anionic semiquinone. With the known structure of medium chain acyl-CoA dehydrogenase, we hypothesize a possible structure for docking the two proteins.
Proc Natl Acad Sci U S A 1996 Dec 10
PMID:Three-dimensional structure of human electron transfer flavoprotein to 2.1-A resolution. 896 55

The enzymic stages of mammalian mitochondrial beta-oxidation were elucidated some 30-40 years ago. However, the discovery of a membrane-associated multifunctional enzyme of beta-oxidation, a membrane-associated acyl-CoA dehydrogenase and characterization of the carnitine palmitoyl transferase system at the protein and at the genetic level has demonstrated that the enzymes of the system itself are incompletely understood. Deficiencies of many of the enzymes have been recognized as important causes of disease. In addition, the study of these disorders has led to a greater understanding of the molecular mechanism of beta-oxidation and the import, processing and assembly of the beta-oxidation enzymes within the mitochondrion. The tissue-specific regulation, intramitochondrial control and supramolecular organization of the pathway is becoming better understood as sensitive analytical and molecular techniques are applied. This review aims to cover enzymological and organizational aspects of mitochondrial beta-oxidation together with the biochemical aspects of inherited disorders of beta-oxidation and the intrinsic control of beta-oxidation.
Biochem J 1996 Dec 01
PMID:Mammalian mitochondrial beta-oxidation. 897 39

Short-chain acyl-CoA dehydrogenase (SCAD) is a homotetrameric mitochondrial flavoenzyme that catalyzes the initial reaction in short-chain fatty acid beta-oxidation. Defects in the SCAD enzyme are associated with failure to thrive, often with neuromuscular dysfunction and elevated urinary excretion of ethylmalonic acid (EMA). To define the genetic basis of SCAD deficiency and ethylmalonic aciduria in patients, we have determined the sequence of the complete coding portion of the human SCAD gene (ACADS) and all of the intron-exon boundaries. The SCAD gene is approximately 13 kb in length and consists of 10 exons. Four polymorphic sites have previously been detected by sequencing of cDNA from fibroblasts of patients excreting elevated amounts of EMA. Three of these polymorphisms (321T/C, 990C/T, 1260G/C) are silent variants, while a 625G/A polymorphism results in an amino acid replacement and has been shown to be associated with ethylmalonic aciduria. From analysis of 18 unrelated Danish families, we show that the four SCAD gene polymorphisms constitute five allelic variants of the SCAD gene, and that the 625A variant together with the less frequent variant form of the three other polymorphisms (321C, 990T, 1260C) constitutes an allelic variant with a frequency of 22% in the general Danish population. Using fluorescence in-situ hybridization, we confirm the localization of the human SCAD gene to the distal part of Chromosome (Chr) 12 and suggest that the SCAD gene is a single-copy gene. The evolutionary relationship between SCAD and five other members of the acyl-CoA dehydrogenase family was investigated by two independent approaches that gave similar phylogenetic trees.
Mamm Genome 1997 Dec
PMID:Structural organization of the human short-chain acyl-CoA dehydrogenase gene. 938 86


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