Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

In a previous paper, we demonstrated that the reductive half-reaction of medium-chain fatty acyl-CoA dehydrogenase (MCAD), utilizing octanoyl-CoA as physiological substrate, generates two (kinetically distinct) forms of the reduced enzyme (MCAD-FADH2) - octenoyl-CoA charge-transfer complexes [Kumar, N.R., & Srivastava, D.K. (1994) Biochemistry 33, 8833-8841]. We present evidence that octenoyl-CoA dissociates from the second (most stable) charge-transfer complex (referred to as CT2) via two alternative ("facile" and "restricted") pathways. The dissociation of octenoyl-CoA via the facile pathway involves the reversal of the overall reductive half-reaction of the enzyme, generating MCAD-FAD - octanoyl-CoA as the Michaelis complex, followed by dissociation of the latter complex into MCAD-FAD + octanoyl-CoA. Hence, via this pathway, octenoyl-CoA is released from the enzyme site in the form of octanoyl-CoA. In contrast, the restricted pathway involves a direct (albeit slow) dissociation of octenoyl-CoA from CT2 to yield MCAD-FADH2 + octenoyl-CoA. The kinetic profile for the dissociation of octenoyl-CoA via the restricted pathway matches the rate of oxidation of the reduced flavin (within CT2) by O2. This suggests that the oxidase activity of the enzyme remains suppressed as long as the reduced enzyme predominates in the form of the charge-transfer complex(es). The oxidase activity of the enzyme emerges concomitantly with the conversion of CT2 to the MCAD-FADH2 - octenoyl-CoA Michaelis complex. The energetic basis for the dissociation of octenoyl-CoA via the facile and restricted pathways and the mechanism of suppression of the oxidase activity of the enzyme are discussed.
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PMID:Facile and restricted pathways for the dissociation of octenoyl-CoA from the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-FADH2-octenoyl-CoA charge-transfer complex: energetics and mechanism of suppression of the enzyme's oxidase activity. 762 13

5,6-Dichloro-4-thia-5-hexenoic acid (DCTH) is a potent hepato- and nephrotoxin that induces mitochondrial dysfunction in rat liver and kidney. Previous studies indicate that DCTH undergoes fatty acid beta-oxidation-dependent bioactivation. The objectives of the present experiments were to elaborate the bioactivation mechanism of DCTH and to examine the interaction of the coenzyme A thioester of DCTH (DCTH-CoA) with the medium-chain acyl-CoA dehydrogenase. In the presence of the terminal electron acceptor ferricenium hexafluorophosphate (FcPF6), DCTH-CoA was oxidized by the medium-chain actyl-CoA dehydrogenase to give 5,6-dichloro-4-thia-trans-2,5-hexadienoyl-CoA. Enoyl-CoA hydratase catalyzed the conversion of 5,6-dichloro-4-thia-trans-2,5-hexadienoyl-CoA to 5,6-dichloro-4-thia-3-hydroxy-5-hexenoyl-CoA, which eliminated 1,2-dichloroethenethiol and gave malonyl-CoA semialdehyde as a product. Chloroacetic acid was detected as a terminal product derived from 1,2-dichloroethenethiol. Incubation of DCTH-CoA with the medium-chain acyl-CoA dehydrogenase in the absence of FcPF6 gave 3-hydroxypropionyl-CoA as the major product and resulted in the irreversible inactivation of the enzyme. Under these conditions, DCTH-CoA apparently undergoes a beta-elimination reaction to give 1,2-dichloroethenethiol and acryloyl-CoA, which is hydrated to give 3-hydroxypropionyl-CoA as the terminal product. The beta-elimination product 1,2-dichloroethenethiol may yield reactive intermediates that inactivate the dehydrogenase. Enzyme inactivation was rapid, DCTH-CoA concentration-dependent, and blocked by octanoyl-CoA, but not by glutathione. The medium-chain acyl-CoA dehydrogenase was not inactivated by acryloyl-CoA, and little inactivation was observed in the presence of FcPF6. These results show that DCTH-CoA is bioactivated by the mitochondrial fatty acid beta-oxidation system to reactive intermediates. This bioactivation mechanism may account for the observed toxicity of DCTH in vivo and in vitro.
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PMID:Medium-chain acyl-CoA dehydrogenase- and enoyl-CoA hydratase-dependent bioactivation of 5,6-dichloro-4-thia-5-hexenoyl-CoA. 770 41

S-2-Br-hexanoyl-CoA and the branched chain isomer S-2-Br-4-methyl-pentanoyl-CoA are affinity labels of the medium-chain acyl-CoA dehydrogenase from pig kidney. The straight chain thioester is both a substrate and an irreversible inhibitor of the dehydrogenase. Inactivation of the enzyme is biphasic and is half-complete in 4 min at pH 6.5, 25 degrees C. Although S-2-Br-hexanoyl-CoA can partially reduce the FAD prosthetic group of the dehydrogenase, inactivation results from attachment of one molecular of inhibitor per subunit of the oxidized enzyme. The branched chain analogue is a very weak substrate of the dehydrogenase (0.1% that of octanoyl-CoA), but is almost as effective an inhibitor of the dehydrogenase. Incubation experiments with [14C]S-2-Br-methyl-pentanoyl-CoA followed by the isolation of radiolabeled peptide show that modification of the active site base, GLU376, is responsible for enzyme inactivation. The data are compatible with a simple nucleophilic attack of the carboxylate base on the C-2 atom of these 2-Br-analogues.
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PMID:S-2-bromo-acyl-CoA analogues are affinity labels for the medium-chain acyl-CoA dehydrogenase from pig kidney. 789 66

Of the different chain length fatty acyl-CoA substrates, octanoyl-CoA has been known as one of the most efficient (and physiological) substrates for the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-catalyzed reaction. The reaction of MCAD-FAD with octanoyl-CoA ([MCAD-FAD] << [octanoyl-CoA]), measured via the stopped-flow technique, at 5 degrees C was characterized by a biphasic decrease and increase in absorptions at 450 and 545 nm, respectively. The average values of the fast (1/tau 1) and slow (1/tau 2) relaxation rate constants, derived from the data at these wavelengths, were found to be 319.7 +/- 33.5 and 28.8 +/- 12.5 s-1, respectively, and both of these relaxation rate constants remained invariant between 8 and 200 microM concentrations of octanoyl-CoA. Under identical experimental conditions, we measured time courses for the interaction of MCAD-FAD with octenoyl-CoA ([MCAD-FAD] << [octenoyl-CoA]) by monitoring the absorption changes at 299, 394, and 440 nm. The binding profile was consistent with a biphasic decrease (at 440 nm) and increase (at 299 and 394 nm) in absorbance, with similar magnitudes of fast [1/tau 1 (average) = 382.3 +/- 39.8 s-1] and slow [1/tau 2 (average) = 14.3 +/- 7.4 s-1] relaxation rate constants. The observed relaxation rate constants were, once again, found to be invariant with changes in the octenoyl-CoA concentration from 40 to 150 microM.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Reductive half-reaction of medium-chain fatty acyl-CoA dehydrogenase utilizing octanoyl-CoA/octenoyl-CoA as a physiological substrate/product pair: similarity in the microscopic pathways of octanoyl-CoA oxidation and octenoyl-CoA binding. 803 75

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.
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PMID:Specific assay of medium-chain acyl-CoA dehydrogenase based on the spectrophotometric measurement of product formation. 810 45

The medium chain acyl-CoA dehydrogenase catalyzes the FAD-dependent oxidation of a variety of acyl-CoA substrates to the corresponding trans-2-enoyl-CoA thioesters. This work identifies 3-methyleneoctanoyl-CoA and 3-methyl-trans-2-octenoyl-CoA as representatives of a new class of mechanism-based inhibitor of the dehydrogenase. One equivalent of either compound generates an inactive reduced flavin species with low absorption at 450 nm and a shoulder at 320 nm suggestive of an N-5 adduct. Reduction is rapid with the 3-methylene analogue (10/s at 1 degree C), but comparatively slow for 3-methyl-trans-2-octenoyl-CoA (1.1 x 10(-4)/s, under the same conditions). The reduced species is very stable, but the adduct can be slowly displaced with a large excess of octanoyl-CoA. The reduced adduct resists oxidation by the facile one-electron oxidant of the dehydrogenase, ferricenium hexafluorophosphate. Evidence that both isomeric inhibitors generate the same reduced flavin species includes an essentially identical visible spectrum, the same kinetics of displacement using octanoyl-CoA, and the same mixture of products on HPLC after denaturation of the treated enzyme with trichloroacetic acid, methanol, or by boiling. Experiments with the corresponding shorter analogues of these inhibitors, 3-methylenebutanoyl-CoA and 3-methyl-2-butenoyl-CoA confirm and extend these findings. These reduced adducts are less stable, allowing the dehydrogenase to catalyze the interconversion of the unconjugated 3-methylenebutanoyl-CoA to the more stable conjugated 3-methyl-2-butenoyl-CoA thioester (Keq ca. 150). These data suggest that alpha-proton abstraction from the 3-methylene derivatives or gamma-proton removal from the 3-methyl-2-enoyl analogues generates a common carbanionic intermediate which attacks oxidized flavin. As would be expected, the unconjugated 3-methylene derivatives are more effective inhibitors of the dehydrogenase than the thermodynamically more stable 3-methylenoyl analogues.
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PMID:3-Methyleneoctanoyl-CoA and 3-methyl-trans-2-octenoyl-CoA: two new mechanism-based inhibitors of medium chain acyl-CoA dehydrogenase from pig kidney. 829 7

Several 2-halo-acyl-CoA derivatives have been synthesized to examine the interaction of these potential inhibitors of mitochondrial beta-oxidation with the purified medium chain acyl-CoA dehydrogenase from pig kidney. Racemic 2-bromo-, 2-chloro-, and 2-fluoro-octanoyl-CoA thioesters show 6.6, 33, and 3.5% of the activity of octanoyl-CoA in the standard assay system, respectively. Only the S-enantiomer of these 2-substituted analogues is a significant substrate of the dehydrogenase, with S-2-bromo-octanoyl-CoA showing a rate of 18% that of octanoyl-CoA, compared to about 1% for the R-isomer. The observations presented here suggest that a detailed understanding of the mode of action of 2-halo-fatty acids as inhibitors of mammalian beta-oxidation will require consideration of the metabolic fate and inhibitory effects of both enantiomers.
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PMID:Stereoselective interaction of 2-halo-acyl-CoA derivatives with medium chain acyl-CoA dehydrogenase from pig kidney. 847 Sep 10

The catalytically essential glutamate residue that initiates catalysis by abstracting the substrate alpha-hydrogen as H+ is located at position 376 (mature MCADH numbering) on loop JK in medium chain acyl-CoA dehydrogenase (MCADH). In long chain acyl-CoA dehydrogenase (LCADH) and isovaleryl-CoA dehydrogenase (IVDH), the corresponding Glu carrying out the same function is placed at position 255 on the adjacent helix G. These glutamates thus act on substrate approaching from two opposite regions at the active center. We have implemented the topology of LCADH in MCADH by carrying out the two mutations Glu376Gly and Thr255Glu. The resulting chimeric enzyme, "medium-/long" chain acyl-CoA dehydrogenase (MLCADH) has approximately 20% of the activity of MCADH and approximately 25% that of LCADH with its best substrates octanoyl-CoA and dodecanoyl-CoA, respectively. MLCADH exhibits an enhanced rate of reoxidation with oxygen, however, with a much narrower substrate chain length specificity that peaks with dodecanoyl-CoA. This is the same maximum as that of LCADH and is thus significantly shifted from that of native MCADH (hexanoyl/octanoyl-CoA). The putative, common ancestor of LCADH and IVDH has two Glu residues, one each at positions 255 and 376. The corresponding MCADH mutant, Thr255Glu (glu/glu-MCADH), is as active as MCADH with octanoyl-CoA; its activity/chain length profile is, however, much narrower. The topology of the Glu as H+ abstracting base seems an important factor in determining chain length specificity and reactivity in acyl-CoA dehydrogenases. The mechanisms underlying these effects are discussed in view of the three-dimensional structure of MLCADH, which is presented in the accompanying paper [Lee et al. (1996) Biochemistry 35, 12412-12420].
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PMID:Medium-long-chain chimeric human Acyl-CoA dehydrogenase: medium-chain enzyme with the active center base arrangement of long-chain Acyl-CoA dehydrogenase. 882 75

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.
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PMID:Oxidative inactivation of a charge transfer complex in the medium-chain acyl-CoA dehydrogenase. 884 70

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.
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PMID:Functional role of the active site glutamate-368 in rat short chain acyl-CoA dehydrogenase. 895 87


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