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)

Recombinant, normal human medium-chain acyl-CoA dehydrogenase (MCADH) and the common, human disease-causing K304E mutant ([Glu304]MCADH) protein were expressed in Escherichia coli using an optimized system, and the enzymes were purified to apparent homogeneity. The crucial factor leading to the production of active [Glu304]MCADH protein is the expression in E. coli cells at reduced temperature (28 degrees C). Expression in the same system at 37 degrees C results in very low amounts of active mutant protein. Several catalytic and physicochemical parameters of these two proteins have been determined and were compared to those of purified pig kidney MCADH. Although [Glu304]MCADH has approximately the same rate of substrate reduction with dodecanoyl-CoA and the same V(max) as human MCADH with the best substrate for the latter, octanoyl-CoA, the K(m) in the mutant MCADH is fourfold higher, which generates a correspondingly lower catalytic efficiency. Importantly, V(max) obtained using the natural acceptor, electron transfer flavoprotein, is only a third that for human MCADH. The V(max)/K(m) versus chain-length profile of the mutant shows a maximum with dodecanoyl-CoA which differs markedly from that of human MCADH, which has maximal efficiency with octanoyl-CoA. The substrate specificity of the mutant is broader with a less pronounced activity peak resembling long-chain acyl-CoA dehydrogenase. The purified mutant enzyme exhibits a reduced thermal stability compared to human wild-type MCADH. The major difference between the two proteins expressed in E. coli is the more pronounced lability of the K304E mutant in crude extracts, which suggests a higher susceptibility to attack by endogenous proteases. Differences between tetrameric [Glu304]MCADH which survives the first step(s) of purification and corresponding MCADH are minor. The overall differences in properties of [Glu304]MCADH together with its impaired folding and tetramer assembly may contribute to the generation of the abnormalities observed in patients homozygous for the K304E mutation.
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PMID:Biochemical characterization of purified, human recombinant Lys304-->Glu medium-chain acyl-CoA dehydrogenase containing the common disease-causing mutation and comparison with the normal enzyme. 920 49

The X-ray crystallographic structure of medium-chain acyl-CoA dehydrogenase (MCAD)-octenoyl-CoA complex reveals that the 3'-phosphate group of CoA is confined to the exterior of the protein structure [approx. 15 A (1.5 nm) away from the enzyme active site], and is fully exposed to the outside solvent environment. To ascertain whether such a distal (3'-phosphate) fragment of CoA plays any significant role in the enzyme catalysis, we investigated the recombinant human liver MCAD (HMCAD)-catalysed reaction by using normal (phospho) and 3'-phosphate-truncated (dephospho) forms of octanoyl-CoA and butyryl-CoA substrates. The steady-state kinetic data revealed that deletion of the 3'-phosphate group from octanoyl-CoA substrate increased the turnover rate of the enzyme to about one-quarter, whereas that from butyryl-CoA substrate decreased the turnover rate of the enzyme to about one-fifth; the Km values of both these substrates were increased by 5-10-fold on deletion of the 3'-phosphate group from the corresponding acyl-CoA substrates. The transient kinetics for the reductive half-reaction, oxidative half-reaction and the dissociation 'off-rate' (of the reaction product from the oxidized enzyme site) were all found to be affected by deletions of the 3'-phosphate group from octanoyl-CoA and butyryl-CoA substrates. A cumulative account of these results reveals that, although the 3'-phosphate group of acyl-CoA substrates might seem 'useless' on the basis of the structural data, it has an essential functional role during HMCAD catalysis.
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PMID:Functional role of a distal (3'-phosphate) group of CoA in the recombinant human liver medium-chain acyl-CoA dehydrogenase-catalysed reaction. 927 Oct 97

A range of 4-thiaacyl-CoA derivatives has been synthesized to study the bioactivation of cytotoxic fatty acids by the mitochondrial medium-chain acyl-CoA dehydrogenase and the peroxisomal acyl-CoA oxidase. Both enzymes catalyze alpha-proton abstraction from normal acyl-CoA substrates with elimination of a beta-hydride equivalent to the FAD prosthetic group. In competition with this oxidation reaction, 4-thiaacyl-CoA thioesters undergo dehydrogenase-catalyzed beta-elimination, providing that the corresponding thiolates are sufficiently good leaving groups and can be accommodated by the active site of the enzyme. Thus, the dehydrogenase catalyzes the elimination of 2-mercaptobenzothiazole and 4-nitrothiophenolate from 4-(2-benzothiazole)-4-thiabutanoyl-CoA and 4-(4-nitrophenyl)-4-thiabutanoyl-CoA, respectively. However, the 2,4-dinitrophenyl-analogue appears too bulky and the unsubstituted thiophenyl-derivative is insufficiently activated for significant elimination. Molecular modeling shows that steric interference from the flavin ring dictates a syn rather than an anti elimination. Acryloyl-CoA, the other product of 4-thiaacyl-CoA elimination reactions, is not a significant inactivator of the medium-chain dehydrogenase. In contrast, the irreversible inactivation observed during beta-elimination using 5,6-dichloro-4-thia-5-hexenoyl-CoA (DCTH-CoA), 5,6-dichloro-7,7,7-trifluoro-4-thia-5-heptenoyl-CoA (DCTFTH-CoA), and 6-chloro-5,5,6-trifluoro-4-thiahexanoyl-CoA (CTFTH-CoA) is caused by release of cytotoxic thiolate products within the active site of the dehydrogenase. The double bond between C5 and C6 found in the vinylic analogues DCTH- and DCTFTH-CoA is not essential for enzyme inactivation, although CTFTH-CoA is a weaker inhibitor of the dehydrogenase. Mechanism-based inactivation with CTFTH-CoA requires elimination, is unaffected by exogenous nucleophiles, and is strongly protected by octanoyl-CoA. The peroxisomal acyl-CoA oxidase efficiently oxidizes 4-thiaacyl-CoA analogues, but is only rapidly inactivated by DCTFTH-CoA. The variable ratio of elimination to oxidation observed for DCTH-, DCTFTH-, and CTFTH-CoA may influence the metabolism of the corresponding cytotoxic alkanoic acids in vivo.
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PMID:Elimination reactions in the medium-chain acyl-CoA dehydrogenase: bioactivation of cytotoxic 4-thiaalkanoic acids. 947 67

The human liver medium chain acyl-CoA dehydrogenase (MCAD)-catalyzed reaction proceeds via abstraction of an alpha-proton from the acyl-CoA substrates by the carboxyl group of Glu-376. By using the methods of site-directed mutagenesis, we replaced Glu-376 by Asp (E376D mutation), expressed the wild-type and mutant enzymes in Escherichia coli, purified them to homogeneity, and compared their kinetic properties. The steady-state kinetic data revealed that the E376D mutation impaired (by about 15-20-fold) the turnover rate of the enzyme as well as its inactivation by 2-octynoyl-CoA. There was no selective solvent deuterium isotope effect on enzyme catalysis. These results lead to the suggestion that the carboxyl group of Asp-376 does not serve as efficient catalytic base as the carboxyl group of Glu-376. The E376D mutation impaired the octanoyl-CoA-dependent reductive half-reaction such that the rate-limiting step of enzyme catalysis shifted from the product dissociation step (in the case of the wild-type enzyme) to the flavin reduction step, and abolished the previously noted kinetic and thermodynamic correspondences between the octanoyl-CoA-dependent reductive half-reaction and the enzyme-octenoyl-CoA interaction [Kumar, N. R., and Srivastava, D. K. (1994) Biochemistry 33, 8833-8841]. Arguments are presented that the Glu-376-->Asp mutation results in uncoupling between the proton transfer and protein conformational change steps during enzyme catalysis.
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PMID:Influence of excision of a methylene group from Glu-376 (Glu376-->Asp mutation) in the medium chain acyl-CoA dehydrogenase-catalyzed reaction. 948 41

The mechanism by which acyl-CoA dehydrogenases initiate catalysis was studied by using p-substituted phenylacetyl-CoAs (substituents-NO2, -CN, and CH3CO-), 3S-C8-, and 3'-dephospho-3S-C8CoA. These analogues lack a beta C-H and cannot undergo alpha,beta-dehydrogenation. Instead they deprotonate at alpha C-H at pH > or = 14 to form delocalized carbanions having strong absorbancies in the near UV-visible spectrum. The pKas of the corresponding phenylacetone analogues were determined as approximately 13.6 (-NO2), approximately 14.5 (-CN), and approximately 14.6 (CH3CO-). Upon binding to human wild-type medium-chain acyl-CoA dehydrogenase (MCADH), all analogues undergo alpha C-H deprotonation. While the extent of deprotonation varies, the anionic products from charge-transfer complexes with the oxidized flavin. From the pH dependence of the dissociation constants (Kd) of p-NO2-phenylacetyl-CoA (4NPA-CoA), 3S-C8-CoA, and 3'-dephospho-3S-C8CoA, four pKas at approximately 5, approximately 6, approximately 7.3, and approximately 8 were identified. They were assigned to the following ionizations: (a) pKa approximately 5, ligand (L-H) in the MCADH approximately ligand complex; (b) pKa approximately 6, Glu376-COOH in uncomplexed MCADH; (c) pKa approximately 7.3, Glu99-COOH in uncomplexed MCADH (Glu99 is a residue that flanks the bottom of the active-center cavity; this pK is absent in the mutant Glu99Gly-MCADH); and (d) pK approximately 8, Glu99-COOH in the MCADH approximately 4NPA-CoA complex. The pKa approximately 6 (b) is not significantly affected in the MCADH approximately 4NPA-CoA complex, but it is increased by > or = 1 pK unit in that with 3S-C8CoA and further in the presence of C8-CoA, the best substrate. The alpha C-H pKas of 4NPA-CoA, of 3S-C8-CoA, and of 3'-dephospho-3S-C8CoA in the complex with MCADH are approximately 5, approximately 5, and approximately 6. Compared to those of the free species these pKa values are therefore lowered by 8 to > or = 11 pH units (50 to > or = 65 kJ mol-1) and are close to the pKa of Glu376-COOH in the complex with substrate/ligand. This effect is ascribed mainly to the hydrogen-bond interactions of the thioester carbonyl group with the ribityl-2'-OH of FAD and Glu376-NH. It is concluded that the pKa shifts induced with normal substrates such as n-octanoyl-CoA are still higher and of the order of 9-13 pK units. With 4NPA-CoA and MCADH, alpha C-H abstraction is fast (kapp approximately 55 s-1 at pH 7.5 and 25 degrees C, deuterium isotope effect approximately 1.34). However, it does not proceed to completion since it constitutes an approach to equilibrium with a finite rate for reprotonation in the pH range 6-9.5. The extent of deprotonation and the respective rates are pH-dependent and reflect apparent pKas of approximately 5 and approximately 7.3, which correspond to those determined in static experiments.
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PMID:Substrate activation by acyl-CoA dehydrogenases: transition-state stabilization and pKs of involved functional groups. 948 10

Following our demonstration that the terminal 3'-phosphate group of acyl-CoA substrates (which is confined to the exterior of the protein structure, and is fully exposed to the outside solvent environment) exhibits a functional role in the recombinant human liver medium-chain acyl-CoA dehydrogenase (MCAD)-catalyzed reaction [Peterson, K. L., and Srivastava, D. K. (1997) Biochem. J. 325, 751-760], we became interested in delineating its thermodynamic contribution in stabilizing the "ground" and "transition" state structures during enzyme catalysis. Since the 3'-phosphate group of the coenzyme A thiolester has the potential to form a hydrogen bond with the side chain group of Asn-191, these studies were performed utilizing both normal and 3'-dephosphorylated forms of octanoyl-CoA and octenoyl-CoA (cumulatively referred to as C8-CoA) as the physiological substrate and product of the enzyme, respectively, as well as utilizing wild-type and Asn191 --> Ala (N191A) site-specific mutant enzymes. The experimental data revealed that the enthalpic contribution of the 3'-phosphate group was similar in both ground and transition states, and was primarily derived from the London-van der Waals interactions (between the 3'-phosphate group of C8-CoA and the surrounding protein moiety), rather than from the potential hydrogen bonding. The temperature dependence of DeltaH degrees for the binding of octenoyl-CoA and 3'-dephosphooctenoyl-CoA revealed that the deletion of the 3'-phosphate group from octenoyl-CoA increased the magnitude of the heat capacity changes (DeltaCp degrees) from -0.53 to -0.59 kcal mol-1 K-1. Although the latter effect could be attributed to an increase in the relative hydrophobicity of the ligand, the experimentally observed DeltaCp degrees's (for either of the ligands) could not be predicted on the basis of the changes in the solvent-accessible surface areas of the enzyme and ligand species. These coupled with the fact that the DeltaCp degrees for the binding of octenoyl-CoA to pig kidney MCAD (which is believed to be structurally identical to human liver MCAD) is only -0.37 kcal mol-1 K-1 [Srivastava, D. K., Wang, S., and Peterson, K. L. (1997) Biochemistry 36, 6359-6366] prompt us to question the reliability of predicting the DeltaCp degrees values of the enzyme-ligand complexes from their X-ray crystallographic data. Arguments are presented that certain intrinisic limitations of the crystallographic data preclude kinetic and thermodynamic predictions about the enzyme-ligand complexes and enzyme catalysis.
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PMID:Thermodynamics of ligand binding and catalysis in human liver medium-chain acyl-CoA dehydrogenase: comparative studies involving normal and 3'-dephosphorylated C8-CoAs and wild-type and Asn191 --> Ala (N191A) mutant enzymes. 973 Aug 39

The activities of beta-oxidation enzymes were measured in extracts of glucose- and triolein-grown cells of Aspergillus niger. Growth on triolein stimulated increased enzyme activity, especially for acyl-CoA dehydrogenase. No acyl-CoA oxidase activity was detected. HPLC analysis after incubation of triolein-grown cell extracts with decanoyl-CoA showed that beta-oxidation was limited to one cycle. Octanoyl-CoA accumulated as the decanoyl-CoA was oxidized. Beta-oxidation enzymes in isolated mitochondrial fractions were also studied. The results are discussed in the context of methyl ketone production by fungi.
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PMID:Oxidation of medium-chain acyl-CoA esters by extracts of Aspergillus niger: enzymology and characterization of intermediates by HPLC. 1020 7

Short-chain acyl-CoA oxidases are beta-oxidation enzymes that are active on short-chain acyl-CoAs and that appear to be present in higher plant peroxisomes and absent in mammalian peroxisomes. Therefore, plant peroxisomes are capable of performing complete beta-oxidation of acyl-CoA chains, whereas mammalian peroxisomes can perform beta-oxidation of only those acyl-CoA chains that are larger than octanoyl-CoA (C8). In this report, we have shown that a novel acyl-CoA oxidase can oxidize short-chain acyl-CoA in plant peroxisomes. A peroxisomal short-chain acyl-CoA oxidase from Arabidopsis was purified following the expression of the Arabidopsis cDNA in a baculovirus expression system. The purified enzyme was active on butyryl-CoA (C4), hexanoyl-CoA (C6), and octanoyl-CoA (C8). Cell fractionation and immunocytochemical analysis revealed that the short-chain acyl-CoA oxidase is localized in peroxisomes. The expression pattern of the short-chain acyl-CoA oxidase was similar to that of peroxisomal 3-ketoacyl-CoA thiolase, a marker enzyme of fatty acid beta-oxidation, during post-germinative growth. Although the molecular structure and amino acid sequence of the enzyme are similar to those of mammalian mitochondrial acyl-CoA dehydrogenase, the purified enzyme has no activity as acyl-CoA dehydrogenase. These results indicate that the short-chain acyl-CoA oxidases function in fatty acid beta-oxidation in plant peroxisomes, and that by the cooperative action of long- and short-chain acyl-CoA oxidases, plant peroxisomes are capable of performing the complete beta-oxidation of acyl-CoA.
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PMID:A novel acyl-CoA oxidase that can oxidize short-chain acyl-CoA in plant peroxisomes. 1021 54

Human 'electron transferring flavoprotein' (ETF) was inactivated by the thiol-specific reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). The kinetic profile showed the reaction followed pseudo-first-order kinetics during the initial phase of inactivation. Monitoring the release of 5-thio-2-nitrobenzoate (TNB) showed that modification of 1 cysteine residue was responsible for the loss of activity. The inactivation of ETF by DTNB could be reversed upon incubation with thiol-containing reagents. The loss of activity was prevented by the inclusion of medium chain acyl-CoA dehydrogenase (MCAD) and octanoyl-CoA. Cyanolysis of the DTNB modified-ETF with KCN led to the release of TNB accompanied presumably by the formation of the thio-cyano enzyme and with almost full recovery of activity. Conservation studies and the lack of 100% inactivation, however, suggested that this cysteine residue is not essential for the interaction with MCAD.
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PMID:5,5'-Dithiobis-(2-nitrobenzoic acid) as a probe for a non-essential cysteine residue at the medium chain acyl-coenzyme A dehydrogenase binding site of the human 'electron transferring flavoprotein' (ETF). 1048 48

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


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