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)

Oxidation of thioester substrates in the medium-chain acyl-CoA dehydrogenase involves alpha-proton abstraction by the catalytic base, Glu376, with transfer of a beta-hydride equivalent to the flavin prosthetic group. Polarization of bound acyl-CoA derivatives by the recombinant human liver enzyme has been studied with 4-thia-trans-2-enoyl-CoA analogues. Polarization is maximal at low pH, with an apparent pK of 9.2 for complexes with the C8 analogue, and progressively lower pK values as the length of the chain increases. This pH effect reflects ionization of the catalytic base, since polarization of a variety of enoyl-CoA analogues by the Glu376Gln mutant is pH independent. Binding of these ligands is accompanied by uptake of about 1 proton with the wild-type enzyme, but only about 0.1 proton with the Glu376Gln mutant. Rapid reaction studies show that proton uptake with the wild-type enzyme occurs at the same rate as polarization of the enoyl-CoA thioester, but is much slower than the initial ligand binding step. Studies with 6-OH-FAD-substituted enzyme show that this isomerization reaction also influences the flavin prosthetic group inducing deprotonation to the green anionic form. The failure of the bound thioether analogue, octyl-SCoA, to elicit pK shifts to flavin and Glu376 shows the importance of the thioester carbonyl oxygen in modulating key properties of the medium-chain enzyme. The role of thioester-mediated desolvation within the active site of the acyl-CoA dehydrogenases is discussed.
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PMID:Protonic equilibria in the reductive half-reaction of the medium-chain acyl-CoA dehydrogenase. 962 95

We investigated the influence of Glu-376-->Asp (E376D) mutation on the UV/visible spectral, thermodynamic, and kinetic properties for the interaction of structurally different types of CoA-ligands (viz., octenoyl-CoA, acetoacetyl-CoA, and indoleacryloyl-CoA) to human liver medium-chain acyl-CoA dehydrogenase (MCAD). Whereas the E376D mutation had minimal/negligible effect on the above properties for the binding of octenoyl-CoA to the enzyme, it had pronounced effects (albeit in opposite directions) for the binding of acetoacetyl-CoA and indoleacryloyl-CoA to the enzyme. In the case of acetoacetyl-CoA, the spectrum of the enzyme-ligand complex (in the charge-transfer region; lambdamax = 545 nm) was 1.8-fold more pronounced, and the DeltaH degrees value for the binding of acetoacetyl-CoA to the enzyme was 5.6 kcal/mol more favorable with wild-type as compared to the E376D mutant enzyme. The kinetic data revealed that the above effects were related to an increase in the dissociation "off-rate" of acetoacetyl-CoA from the enzyme-acetoacetyl-CoA complex. In contrast, in the case of IACoA, the resultant UV/visible spectrum of the enzyme-IACoA complex (lambdamax = 416 nm) was 2.7-fold less pronounced, and the DeltaH degrees value of the enzyme-IACoA complex was 6.4 kcal/mol less favorable with the wild-type than the E376D mutant enzyme. The latter effects were supported by the fact that the above mutation impaired the dissociation "off-rate" of IACoA from the enzyme-IACoA complex by 5.7-fold. Molecular model building studies revealed that the discriminatory influence of the E376D mutation on the spectral, thermodynamic, and kinetic properties of the enzyme-ligand complexes is due to ligand-specific changes in the spatial relationship between the FAD and CoA-ligands at the enzyme site. Arguments are presented that the "void" created by excision of a methylene group from Glu-376 (upon Glu-376-->Asp mutation) is adjusted differently upon interaction with structurally different types of CoA-ligands.
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PMID:Discriminatory influence of Glu-376-->Asp mutation in medium-chain acyl-CoA dehydrogenase on the binding of selected CoA-ligands: spectroscopic, thermodynamic, kinetic, and model building studies. 962 96

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

The mechanism underlying the recognition and activation of the substrate for medium-chain acyl-CoA dehydrogenase (MCAD) was spectroscopically investigated using 3-thiaacyl-CoAs as substrate analogs. The complex of MCAD with 3-thiaoctanoyl-CoA (3-thia-C8-CoA) exhibited a charge-transfer (CT) band with a molar extinction coefficient of epsilon808 = 9.1 mM-1.cm-1. With increasing 3-thiaacyl-chain length, the CT-band intensity of the complex decreased concomitantly with changes in the FAD absorption at 416 and 482 nm, and no CT band was detected in complexes with chain-lengths longer than C15. Detailed analysis of the absorption spectra suggested that the complexed states represent a two-state equilibrium between the CT-inducing form and the CT-non-inducing form. 13C-NMR measurements with 13C-labeled ligand clarified that 3-thia-C8-CoA is complexed to MCAD in an anionic form with signals detected at 163.7 and 101.2 ppm for 13C(1) and 13C(2), respectively. In the MCAD complex with 13C(1)-labeled 3-thia-C12-CoA, two signals for the bound ligand were observed at 163.7 and 198.3 ppm, and assigned to the anionic and neutral forms, respectively. Only the neutral form signal was measured at 200.6 ppm in the complex with 13C(1)-labeled 3-thia-C17-CoA. These results indicate that the CT band can be explained in terms of an internal equilibrium between anionic (CT-inducing) and neutral (CT-non-inducing) forms of the bound ligand. Resonance Raman spectra of the MCAD.3-thia-C8-CoA complex, with excitation at the CT band, showed enhanced bands, among which the 854- and 1,368-cm-1 bands were assigned to the S-C(2) stretching mode of the ligand and to flavin band VII, respectively. Since the enhanced bands were observed at the same wave numbers in complexes with C8, C12, and C14-ligands, it appears that the CT-inducing form shares a common alignment relative to oxidized flavin irrespective of differences in the acyl-chain length. However, with longer ligands, the degree of resonance enhancement of the Raman bands decreased in parallel with the CT-band intensity; this is compatible with the increase in the CT-non-inducing form in complexes with longer ligands. Furthermore, the pH dependence of the CT band gave an apparent pKa = 5.6-5.7 for ligands with chain-lengths of C8-C12. The NMR measurements revealed that, like chain-length dependence, the pH dependence can be explained by a two-state equilibrium derived from the protonation/deprotonation of the CT-inducing form of the bound ligand. On the basis of these results we have established a novel model to explain the mechanism of recognition and activation of the substrates/ligands by MCAD.
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PMID:Mechanism for the recognition and activation of substrate in medium-chain acyl-CoA dehydrogenase. 999 Jan 25

The crystal structure of electron transfer flavoprotein (ETF) from Paracoccus denitrificans was determined and refined to an R-factor of 19.3% at 2.6 A resolution. The overall fold is identical to that of the human enzyme, with the exception of a single loop region. Like the human structure, the structure of the P. denitrificans ETF is comprised of three distinct domains, two contributed by the alpha-subunit and the third from the beta-subunit. Close analysis of the structure reveals that the loop containing betaI63 is in part responsible for conferring the high specificity of AMP binding by the ETF protein. Using the sequence and structures of the human and P. denitrificans enzymes as models, a detailed sequence alignment has been constructed for several members of the ETF family, including sequences derived for the putative FixA and FixB proteins. From this alignment, it is evident that in all members of the ETF family the residues located in the immediate vicinity of the FAD cofactor are identical, with the exception of the substitution of serine and leucine residues in the W3A1 ETF protein for the human residues alphaT266 and betaY16, respectively. Mapping of ionic differences between the human and P. denitrificans ETF onto the structure identifies a surface that is electrostatically very similar between the two proteins, thus supporting a previous docking model between human ETF and pig medium-chain acyl-CoA dehydrogenase (MCAD). Analysis of the ionic strength dependence of the electron transfer reaction between either human or P. denitrificans ETF and MCAD demonstrates that the human ETF functions optimally at low ( approximately 10 mequiv) ionic strength, while P. denitrificans ETF is a better electron acceptor at higher (>75 mequiv) ionic strength. This suggests that the electrostatic surface potential of the two proteins is very different and is consistent with the difference in isoelectric points between the proteins. Analysis of the electrostatic potentials of the human and P. denitrificans ETFs reveals that the P. denitrificans ETF is more negatively charged. This excess negative charge may contribute to the difference in redox potentials between the two ETF flavoproteins and suggests an explanation for the opposing ionic strength dependencies for the reaction of MCAD with the two ETFs. Furthermore, by analysis of a model of the previously described human-P. denitrificans chimeric ETF protein, it is possible to identify one region of ETF that participates in docking with ETF-ubiquinone oxidoreductase, the physiological electron acceptor for ETF.
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PMID:Crystal structure of Paracoccus denitrificans electron transfer flavoprotein: structural and electrostatic analysis of a conserved flavin binding domain. 1002 81

Electron-transfer flavoprotein (ETF) serves as an intermediate electron carrier between primary flavoprotein dehydrogenases and terminal respiratory chains in mitochondria and prokaryotic cells. The three-dimensional structures of human and Paracoccus denitrificans ETFs determined by X-ray crystallography indicate that the 4'-hydroxyl of the ribityl side chain of FAD is hydrogen bonded to N(1) of the flavin ring. We have substituted 4'-deoxy-FAD for the native FAD and investigated the analog-containing ETF to determine the role of this rare intra-cofactor hydrogen bond. The binding constants for 4'-deoxy-FAD and FAD with the apoprotein are very similar, and the energy of binding differs by only 2 kJ/mol. The overall two-electron oxidation-reduction potential of 4'-deoxy-FAD in solution is identical to that of FAD. However, the potential of the oxidized/semiquinone couple of the ETF containing 4'-deoxy-FAD is 0.116 V less than the oxidized/semiquinone couple of the native protein. These data suggest that the 4'-hydoxyl-N(1) hydrogen bond stabilizes the anionic semiquinone in which negative charge is delocalized over the N(1)-C(2)O region. Transfer of the second electron to 4'-deoxy-FAD reconstituted ETF is extremely slow, and it was very difficult to achieve complete reduction of the flavin semiquinone to the hydroquinone. The turnover of medium chain acyl-CoA dehydrogenase with native ETF and ETF containing the 4'-deoxy analogue was essentially identical when the reduced ETF was recycled by reduction of 2,6-dichlorophenolindophenol. However, the steady-state turnover of the dehydrogenase with 4'-deoxy-FAD was only 23% of the turnover with native ETF when ETF semiquinone formation was assayed directly under anaerobic conditions. This is consistent with the decreased potential of the oxidized semiquinone couple of the analog-containing ETF. ETF containing 4'-deoxy-FAD neither donates to nor accepts electrons from electron-transfer flavoprotein ubiquinone oxidoreductase (ETF-QO) at significant rates (</=0.5% the wild-type rates). These results indicate that the 4'-hydroxyl-N(1) hydrogen bond plays a major role in the stabilization of the anionic semiquinone and anionic hydroquinone oxidation states of ETF and that this hydrogen bond may provide a pathway for electron transfer between the ETF flavin and the flavin of ETF-QO.
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PMID:The intraflavin hydrogen bond in human electron transfer flavoprotein modulates redox potentials and may participate in electron transfer. 1042 53

We previously reported that the kinetic profiles for the association and dissociation of functionally diverse C(8)-CoA-ligands, viz., octanoyl-CoA (substrate), octenoyl-CoA (product), and octynoyl-CoA (inactivator) with medium chain acyl-CoA dehydrogenase (MCAD), were essentially identical, suggesting that the protein conformational changes played an essential role during ligand binding and/or catalysis [Peterson, K. L., Sergienko, E. E., Wu, Y., Kumar, N. R., Strauss, A. W., Oleson, A. E., Muhonen, W. W., Shabb, J. B., and Srivastava, D. K. (1995) Biochemisry 34, 14942-14953]. To ascertain the structural basis of the above similarity, we investigated the kinetics of association and dissociation of alpha-CH-->NH-substituted C(8)-CoA, namely, 2-azaoctanoyl-CoA, with the recombinant form of human liver MCAD. The rapid-scanning and single wavelength stopped-flow data for the binding of 2-azaoctanoyl-CoA to MCAD revealed that the overall interaction proceeds via two steps. The first (fast) step involves the formation of an enzyme-ligand collision complex (with a dissociation constant of K(c)), followed by a slow isomerization step (with forward and reverse rate constants of k(f) and k(r), respectively) with concomitant changes in the electronic structure of the enzyme-bound FAD. Since the latter step involves a concurrent change in the enzyme's tryptophan fluorescence, it is suggested that the isomerization step is coupled to the changes in the protein conformation. Although the overall binding affinity (K(d)) of the enzyme-2-azaoctanoyl-CoA complex is similar to that of the enzyme-octenoyl-CoA complex, their microscopic equilibria within the collision and isomerized complexes show an opposite relationship. These results coupled with the isothermal titration microcalorimetric studies lead to the suggestion that the electrostatic interaction within the enzyme site phase modulates the microscopic steps, as well as their corresponding ground and transition states, during the course of the enzyme-ligand interaction.
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PMID:Influence of alpha-CH-->NH substitution in C8-CoA on the kinetics of association and dissociation of ligands with medium chain acyl-CoA dehydrogenase. 1102 46

The redox-inactive thioester analog 3-thia-octanoyl-CoA blocks transfer of a hydride equivalent to the flavin prosthetic group of the medium-chain acyl-CoA dehydrogenase with the accumulation of a stable enolate intermediate not encountered with normal substrates. Substitution of the normal flavin with 5-deaza-FAD would thus be expected to lead to enolate formation with both normal and 3-thia-substrate analogs, because reduction of the 5-deaza-enzyme is thermodynamically highly unfavorable. However, spectrophotometric titrations show that neither ligand forms significant enolate species with the 5-deaza-FAD enzyme. Similarly, the substituted dehydrogenase catalyzes undetectable alpha-proton exchange with octanoyl-CoA and ca. 1% of the corresponding rate with 3-thia-octanoyl-CoA when compared to the native enzyme. This inability to stabilize enolate species is not simply due to impaired binding of CoA-thioester analogs, because binding of a range of ligands is weakened by only 2- to 10-fold with the 5-deaza-enzyme. 4-Thia-trans-2-enoyl-CoA product is polarized normally on binding to the substituted protein, showing that this critical aspect of catalysis is apparently normal. These data, together with studies with CoA-persulfide and acetoacetyl- and p-nitrophenylacetyl-CoA, suggest that 5-deaza-FAD substitution exerts subtle, unanticipated, effects on the reductive half-reaction of the medium-chain acyl-CoA dehydrogenase. The involvement of charge-transfer interactions in the acidification of weakly acidic acyl-CoA thioesters is discussed.
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PMID:Thioester enolate stabilization in the acyl-CoA dehydrogenases: the effect of 5-deaza-flavin substitution. 1148 11

Isovaleryl-CoA dehydrogenase (IVD) is a homotetrameric flavoenzyme, which catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA and transfers electrons to the electron-transferring flavoprotein, and is a member of the acyl-CoA dehydrogenase (ACD) enzyme family. Human IVD crystal structure with a bound substrate analogue shows the guanidino group of Arg387, a conserved residue among other members of the ACD enzyme family, juxtaposed to a phosphate oxygen of the 4'-phosphopantothiene moiety of the substrate analogue. Site-directed mutagenesis was used to investigate the role of Arg387 in substrate binding and enzyme function. Replacing this residue with Lys, Ala, Gln, or Glu resulted in stable proteins. Spectrophotometric substrate binding assays indicated that the Arg387Lys mutant was able to form the charge-transfer complex intermediate with similar efficiency to wild type, while the rest of the mutants were significantly less able to properly form this intermediate. However, the Km of the isovaleryl-CoA for the Arg387Lys mutant was 20.3 compared to 1.5 microM for the wild type. The Km for the rest of the mutants were 75.6, 195, and 550 microM, respectively. The catalytic efficiency per mole of FAD was 20.3, 3.3, 2.0, and 0.34 for the mutants, respectively, compared to 260 microM(-1) x min(-1) for the wild type. These results substantiate the important role of Arg387 in anchoring the substrate, and are consistent with the hypothesis that residues distant from the active site are important for stabilizing the enzyme:substrate/product complex, and could play an important role in the mechanism of the enzyme-catalyzed reaction.
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PMID:Arginine 387 of human isovaleryl-CoA dehydrogenase plays a crucial role in substrate/product binding. 1159 19

The flavoprotein nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to the respective aldehydes with production of nitrite and hydrogen peroxide. The sequences of several peptides from the fungal enzyme were used to design oligonucleotides for the isolation of a portion of the NAO gene from an F. oxysporum genomic DNA preparation. This sequence was used to clone the cDNA for NAO from an F. oxysporum cDNA library. The sequence of the cloned cDNA showed that NOA is a member of the acyl-CoA dehydrogenase (ACAD) superfamily. The members of this family share with NAO a mechanism that is initiated by proton removal from carbon, suggesting a common chemical reaction for this superfamily. NAO was expressed in Escherichia coli and the recombinant enzyme was characterized. Recombinant NAO has identical kinetic parameters to enzyme isolated from F. oxysporum but is isolated with oxidized FAD rather than the nitrobutyl-FAD found in the fungal enzyme. NAO purified from E. coli or from F. oxysporum has no detectable ACAD activity on short- or medium-chain acyl CoAs, and medium-chain acyl-CoA dehydrogenase and short-chain acyl-CoA dehydrogenase are unable to catalyze oxidation of nitroalkanes.
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PMID:Cloning of nitroalkane oxidase from Fusarium oxysporum identifies a new member of the acyl-CoA dehydrogenase superfamily. 1186 31


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