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:1.3.1.8 (acyl-CoA dehydrogenase)
785 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Reduction of the oxidized FAD at the active site of porcine liver fatty acyl-CoA dehydrogenase by butyryl-CoA results in bleaching of 30 to 60% of the 450 nm absorbance of flavin and in the production of a new absorbance band at 565 nm. The wavelength of the maximum absorbance of this new band (lambda max) is dependent on the chemical nature of the substrate, e.g. this band occurs at 645 nm when beta-2-furylpropionyl-CoA (a pseudosubstrate) reacts with enzyme. Since lambda max for this band is substrate-dependent, the band is most likely the result of charge transfer complex formation between oxidized fatty acyl-CoA, and the reduced flavin of the enzyme. The rate profile for the reaction of butyryl-CoA and enzyme is biphasic at 450 nm but consists of a single exponential process at 565 nm. The rate constant for reaction at 565 nm is approximately 12 s-1 ((butyryl-CoA) = 2.5 x 10(-5) M, pH 8.6), and the 450 nm rate profile can be fit to a rate equation for two sequential reactions of rate constant 12 s-1 and 3.4 s-1, the amount of flavin reduction in each kinetic step being approximately 50%. The deuterium isotope effect measured on each step of the biphasic time course of the 450 nm reaction is very large, in the range kH/kD = 30 to 50. The rate profile at 565 nm for perdeuterobutyryl-CoA is markedly different than that for the protiobutyryl-CoA in that it is biphasic. It appears that two rate processes have been separated by virtue of different isotope effects; the first process shows kH/kD = 2 while the second shows kH/kD = 50. The data are interpreted in terms of a mechanism involving an obligatory charge transfer complex.
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PMID:The deuterium isotope effect upon the reaction of fatty acyl-CoA dehydrogenase and butyryl-CoA. 741 Apr 13

The flavoprotein pig kidney general acyl-CoA dehydrogenase contains a single catalytically essential methionine residue/FAD which reacts with iodoacetate at pH 6.6. S-Carboxymethylation of this residue generates an inactive enzyme derivative which retains FAD and the tetrameric structure of the native protein. The derivative binds actanoyl-CoA and palmityol-CoA with concomitant perturbation of the flavin chromophore, but the characterisitic spectrum of the reduced enzyme-enoyl-CoA complex is not observed. In addition, octanyol-CoA strongly protects the native enzyme against alkylation with iodoacetate. These results suggest that the methionine residue is within the active center of acyl-CoA dehydrogenase. Carboxymethylation of this residue may disrupt the precise orientation of the substrate required to achieve transfer of reducing equivalents to the flavin. Pig kidney general acyl-CoA dehydrogenase does not contain exposed catalytically essential cysteine residues.
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PMID:An essential methionine in pig kidney general acyl-CoA dehydrogenase. 745 27

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

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

We have investigated the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-catalyzed reaction via rapid-scanning stopped-flow (RSSF) UV/vis spectroscopy, combined with the single-wavelength stopped-flow technique, utilizing 3-indolepropionyl-CoA (IPCoA) and trans-3-indoleacryloyl-CoA (IACoA) as chromophoric pseudosubstrates. The RSSF spectral data reveal that formation of an intermediary species with an absorbance maximum at 400 nm and a broad charge-transfer band around 600 nm accompanies the reduction of MCAD-FAD by IPCoA. In the presence of high concentrations of enzyme ([MCAD] >> [IPCoA]) the intermediary spectral band at 400 nm remains unperturbed, whereas in the presence of low concentrations of enzyme ([MCAD] << [IPCoA]) it slowly shifts to an absorption band with an absorbance maximum at 370 nm. Appearance and disappearance of this intermediary species coincides with the appearance and disappearance of the charge-transfer band. Single-wavelength stopped-flow studies, performed under similar high and low enzyme conditions, were consistent with one (1/tau 1) and two (1/tau 1 > 1/tau 2) relaxation rate constants, respectively. These findings, combined with relaxation studies performed in the reverse directions as well as substrate and product binding studies with the oxidized and reduced forms of the enzyme, have allowed us to conclude the following: (1) the intermediary species possesses the properties of reduced flavin and highly conjugated reaction product IACoA (absorbance maximum = 400 nm); (2) this intermediary species collapses into an MCAD-FADH2-IACoA complex (absorbance maximum = 370 nm) in the presence of excessive concentrations of IPCoA; the collapse is being driven by the competitive binding of IPCoA with the reduced form of the enzyme; (3) the 400-nm absorption band and the charge-transfer band are given by the same intermediary species formed during the enzyme-catalyzed reaction pathway. The role of protein conformational changes in modulating the substrate/product structures during the MCAD-catalyzed reaction is discussed.
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PMID:Detection and identification of a chromophoric intermediate during the medium-chain fatty acyl-CoA dehydrogenase-catalyzed reaction via rapid-scanning UV/visible spectroscopy. 826 94

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

Threonine 244 in the alpha subunit of Paracoccus denitrificans transfer flavoprotein (ETF) lies seven residues to the amino terminus of a proposed dinucleotide binding motif for the ADP moiety of the FAD prosthetic group. This residue is highly conserved in the alpha subunits of all known ETFs, and the most frequent pathogenic mutation in human ETF encodes a methionine substitution at the corresponding position, alphaT266. The X-ray crystal structures of human and P. denitrificans ETFs are very similar. The hydroxyl hydrogen and a backbone amide hydrogen of alphaT266 are hydrogen bonded to N(5) and C(4)O of the flavin, respectively, and the corresponding alphaT244 has the same structural role in P. denitrificans ETF. We substituted a methionine for T244 in the alpha subunit of P. denitrificans ETF and expressed the mutant ETF in Escherichia coli. The mutant protein was purified, characterized, and compared with wild type P. denitrificans ETF. The mutation has no significant effect on the global structure of the protein as inferred from visible and near-ultraviolet absorption and circular dichroism spectra, far-ultraviolet circular dichroism spectra, and infrared spectra in 1H2O and 2H2O. Intrinsic fluorescence due to tryptophan of the mutant protein is 60% greater than that of the wild type ETF. This increased tryptophan fluorescence is probably due to a change in the environment of the nearby W239. Tyrosine fluorescence is unchanged in the mutant protein, although two tyrosine residues are close to the site of the mutation. These results indicate that a change in structure is minor and localized. Kinetic constants of the reductive half-reaction of ETF with porcine medium chain acyl-CoA dehydrogenase are unaltered when alphaT244M ETF serves as the substrate; however, the mutant ETF fails to exhibit saturation kinetics when the semiquinone form of the protein is used as the substrate in the disproportionation reaction catalyzed by P. denitrificans electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). The redox behavior of the mutant ETF was also altered as determined from the equilibrium constant of the disproportionation reaction. The separation of flavin redox potentials between the oxidized/semiquinone couple and semiquinone/hydroquinone couple are -6 mV in the wild type ETF and -27 mV in the mutant ETF. The mutation does not alter the AMP content of the protein, although the extent and fidelity of AMP-dependent, in vitro renaturation of the mutant AMP-free apoETF is reduced by 57% compared to renaturation of wild type apoETF, likely due to the absence of the potential hydrogen bond donor T244.
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PMID:alphaT244M mutation affects the redox, kinetic, and in vitro folding properties of Paracoccus denitrificans electron transfer flavoprotein. 910 14

The catalytically essential glutamate base in the acyl-CoA dehydrogenase family is found either on the loop between J and K helices (e.g., in short-chain, medium-chain, and glutaryl-CoA dehydrogenases) or on the G helix (long-chain and isovaleryl-CoA dehydrogenases). While active-site bases at either position are functionally equivalent with respect to alpha-proton abstraction, reactions that require removal of a gamma-proton show marked differences between the two enzyme classes. Thus short-chain, medium-chain, and glutaryl-CoA dehydrogenase are rapidly inactivated by 2-pentynoyl-CoA with abstraction of a gamma-proton, whereas isovaleryl-CoA dehydrogenase is not significantly inhibited. This resistance is not due to weak binding: the complex between isovaleryl-CoA dehydrogenase and 2-pentynoyl-CoA shows a Kd of 1.8 microM at pH 7.6. Migration of the catalytic base to the loop between J and K helices (using the Glu254Gly/Ala375Glu double mutant) makes isovaleryl-CoA dehydrogenase sensitive to irreversible inhibition by 2-pentynoyl-CoA. Molecular modeling suggests that this mutation brings the catalytic base close enough to abstract a gamma-proton from the bound inhibitor. Experiments with two mechanism-based inactivators that target the FAD of the medium- and short-chain acyl-CoA dehydrogenases support this conclusion. 3-Methyl-3-butenoyl-CoA requires activation by alpha-proton abstraction and rapidly yields a reduced flavin adduct with wild-type isovaleryl-CoA dehydrogenase. In contrast, the isomeric 3-methyl-2-butenoyl-CoA is inert toward this enzyme because it cannot be activated by gamma-proton abstraction. Molecular modeling supports these observations. This unusual selectivity toward mechanism-based inactivators provides additional discrimination between members of the acyl-CoA dehydrogenase family.
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PMID:Mechanism-based inhibitor discrimination in the acyl-CoA dehydrogenases. 920 18

Mature medium chain acyl-CoA dehydrogenase isolated from pig kidney (pkMCADH) and originating from mitochondria carries a phosphate group as demonstrated by 31P-NMR-spectroscopy and chemical analysis. Two broad resonances at -6.3 and -8 ppm are observed and are assigned to the pyrophosphate group of the cofactor FAD. A third, narrow resonance at 4.65 ppm indicates the presence of a phosphomonoester residue. Chemical analysis of intact pkMCADH shows the presence of 3 +/- 0.3 phosphates, those of FAD and of an additional covalently attached phosphate. With recombinant, human wild type MCADH expressed in and purified from E. coli only the two FAD phosphates (2 +/- 0.35) are found. Similarly, pkMCADH which has been converted to the apoenzyme and reconstituted to holoenzyme also contains 2 +/- 0.4 phosphates. The covalently bound phosphate can be hydrolyzed by phosphatase and subsequently removed by dialysis. The phosphate group has no detectable effect on the catalytic activity of the MCADH measured with artificial and natural electron acceptors such as pig electron transferring flavoprotein. However, phosphorylation has a marked effect on protein solubility which is +5-fold lower for the dephosphorylated protein.
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PMID:Medium-chain acyl CoA dehydrogenase: evidence for phosphorylation. 942 98

We studied the roles of Thr-136 (T136) and Glu-137 (E137) in the biogenesis of medium chain acyl-CoA dehydrogenase (MCAD) by altering the former to Ser (T136S), Asp (T136D), or Leu (T136L) and the latter to Asp (E137D), Gln (E137Q), or Lys (E137K). After import into mitochondria, T136S and E137D were assembled into the native tetramer as efficiently as the wild-type. The tetrameric assembly of four other variants with a nonconservative substitution was severely impaired. When expressed in Escherichia coli as the mature subunit, the amounts of the catalytically active forms of T136S and E137D were comparable to wild-type, whereas four nonconservative variants were lost as aggregates. Of these nonconservative variants, only T136D formed catalytically active tetramer when the culture broth and buffers were supplemented with riboflavin and FAD, respectively. Culturing T136L or E137K at a lower temperature (28 degreesC) did not increase the yield at all, suggesting the severity of disruption of biogenesis. These results, together with the previous crystallographic findings, indicate that the T136 hydroxyl is a major FAD-binding site, and that E137 carboxyl plays a key role in the beta-domain folding, through salt bridge formation with K164. These findings also support the notion that the isoalloxazine ring plays a critical role in the MCAD folding, presumably exerting nucleating effects.
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PMID:The roles of threonine-136 and glutamate-137 of human medium chain acyl-CoA dehydrogenase in FAD binding and peptide folding using site-directed mutagenesis: creation of an FAD-dependent mutant, T136D. 975 Jan 63


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