3-Methylene-octanoyl-CoA and 3-methyl-trans-2-octenoyl-CoA: Two new mechanism-based inhibitors of medium chain acyl-CoA dehydrogenase from pig kidney

Cummings, J G; Thorpe, Colin

doi:10.1021/bi00169a021

Cited by 24 publications

(47 citation statements)

References 33 publications

(57 reference statements)

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“…4C). Such a difference between the absorbance and CD spectra suggests a difference in the conformational shift of the reduced form of enzyme when it complexed with different substrates (25,27,36,52). This could be due to alterations in the conformation of the flavin isoalloxazine ring itself or in its position relative to an amino acid residue that can alter the flavin signal.…”

Section: Kinetic Properties Of Purified Wild Type and Mutantmentioning

confidence: 99%

“…Most frequently, the interaction between an ACD and various substrates is studied by monitoring characteristic absorbance changes at 450 and 560 nm following the addition of substrate to enzyme under anaerobic conditions. A decrease in absorbance at 450 nm is characteristic of the reduction of the essential FAD coenzyme, whereas an increase in absorbance at 560 nm is because of a new resonance signal related to the charge-transfer complex in the enzyme reaction (1,4,(25)(26)(27). In some situations, however, the observed changes at these wavelengths have been multiphasic, leading to difficulty in interpretation of the results.…”

mentioning

confidence: 99%

“…This has been postulated to be due to excess substrate:enzyme ratios in the reaction mixture as shown in Scheme 2 (26). Furthermore, the change in the 560 nm maximum is reduced or absent when non-optimum substrates interact with an ACD (27). To circumvent some of these difficulties, we have developed a real time surface plasmon resonance (SPR) method to measure the overall binding affinity of SBCAD for its natural substrate at low substrate concentrations.…”

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confidence: 99%

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A Novel Approach to the Characterization of Substrate Specificity in Short/Branched Chain Acyl-CoA Dehydrogenase

Burghardt

Vockley

2003

Journal of Biological Chemistry

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Rat and human short/branched chain acyl-CoA dehydrogenases exhibit key differences in substrate specificity despite an overall amino acid identity of 85% between them. Rat short/branched chain acyl-CoA dehydrogenases (SBCAD) are more active toward substrates with longer carbon side chains than human SBCAD, whereas the human enzyme utilizes substrates with longer primary carbon chains. The mechanism underlying this difference in substrate specificity was investigated with a novel surface plasmon resonance assay combined with absorbance and circular dichroism spectroscopy, and kinetics analysis of wild type SBCADs and mutants with altered amino acid residues in the substrate binding pocket. Results show that a relatively few amino acid residues are critical for determining the difference in substrate specificity seen between the human and rat enzymes and that alteration of these residues influences different portions of the enzyme mechanism. Molecular modeling of the SBCAD structure suggests that position 104 at the bottom of the substrate binding pocket is important in determining the length of the primary carbon chain that can be accommodated. Conformational changes caused by alteration of residues at positions 105 and 177 directly affect the rate of electron transfer in the dehydrogenation reactions, and are likely transmitted from the bottom of the substrate binding pocket to ␤-sheet 3. Differences between the rat and human enzyme at positions 383, 222, and 220 alter substrate specificity without affecting substrate binding. Modeling predicts that these residues combine to determine the distance between the flavin ring of FAD and the catalytic base, without changing the opening of the substrate binding pocket.The acyl-CoA dehydrogenases (ACDs) 1 are a family of related enzymes that catalyze the ␣, ␤-dehydrogenation of acylCoA esters, transferring electrons to electron transferring flavoprotein (1-4). Deficiencies of these enzymes are important causes of human disease (3-7). Biochemical and immunological studies have identified at least 9 distinct members of this enzyme family, each having a characteristic substrate utilization pattern (8 -13). Very long, long, medium, and short chain acyl-CoA dehydrogenases (VLCAD, LCAD, MCAD, and SCAD, respectively) catalyze the first step in the mitochondrial ␤-oxidation of fatty acids with substrate optima of 16, 16, 8, and 4 carbon chains, respectively (3, 14 -15). A new ACD has been identified recently (ACAD9) that is also active against long chain acyl-CoA substrates (16). Isovaleryl-CoA dehydrogenase (IVD), short/branched chain acyl-CoA dehydrogenase (SBCAD, also known as 2-methyl branched chain acyl-CoA dehydrogenase), and isobutyryl-CoA dehydrogenase (IBD) catalyze the third step in leucine, isoleucine, and valine metabolism, respectively (2-4, 11, 12, 17, 18), whereas glutaryl-CoA dehydrogenase is active in lysine metabolism (19).We have shown previously that SCAD, SBCAD, and IBD can all utilize butyryl-CoA as substrate (albeit with different efficiencies), whereas ...

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Section: Kinetic Properties Of Purified Wild Type and Mutantmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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A Novel Approach to the Characterization of Substrate Specificity in Short/Branched Chain Acyl-CoA Dehydrogenase

Burghardt

Vockley

2003

Journal of Biological Chemistry

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“…To determine whether SCAD required a substrate bound form to interact with ETF, one of two substrate analogues were incubated with SCAD. Methylenecyclopropylacetyl-CoA is a specific acyl-CoA dehydrogenase inhibitor that covalently modifies the catalytic base upon entering the substrate-binding pocket (50,51). Acetoacetyl-CoA is a dead end inhibitor that is thought to mimic the charge-transfer intermediate state of substrate binding (52).…”

Section: Discussionmentioning

confidence: 99%

Mammalian Electron Transferring Flavoprotein·Flavoprotein Dehydrogenase Complexes Observed by Microelectrospray Ionization-Mass Spectrometry and Surface Plasmon Resonance

Hoard‐Fruchey

Goetzman

Benson

et al. 2004

Journal of Biological Chemistry

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Microelectrospray ionization-mass spectrometry was used to directly observe electron transferring flavoprotein⅐flavoprotein dehydrogenase interactions. When electron transferring flavoprotein and porcine dimethylglycine dehydrogenase or sarcosine dehydrogenase were incubated together in the absence of substrate, a relative molecular mass corresponding to the flavoprotein⅐electron transferring flavoprotein complex was observed, providing the first direct observation of these mammalian complexes. When an acyl-CoA dehydrogenase family member, human short chain acyl-CoA dehydrogenase, was incubated with dimethylglycine dehydrogenase and electron transferring flavoprotein, the microelectrospray ionization-mass spectrometry signal for the dimethylglycine dehydrogenase⅐electron transferring flavoprotein complex decreased, indicating that the acyl-CoA dehydrogenases have the ability to compete with the dimethylglycine dehydrogenase/sarcosine dehydrogenase family for access to electron transferring flavoprotein. Surface plasmon resonance solution competition experiments revealed affinity constants of 2.0 and 5.0 M for the dimethylglycine dehydrogenaseelectron transferring flavoprotein and short chain acyl-CoA dehydrogenase-electron transferring flavoprotein interactions, respectively, suggesting the same or closely overlapping binding motif(s) on electron transferring flavoprotein for dehydrogenase interaction.Mass spectrometry and surface plasmon resonance are proving valuable techniques for functional proteomics, because each provides powerful detection and identification capabilities for assessing in vivo protein-protein interactions (1). In this work, the interactions between electron transferring flavoprotein (ETF) 1 and two flavoprotein dehydrogenase families are assessed by microelectrospray ionization-mass spectrometry and surface plasmon resonance, demonstrating the complementarity of the two techniques.ETF is a mitochondrial matrix electron acceptor that interacts with and accepts electrons from at least 10 flavoprotein dehydrogenases (2). ETF donates these electrons to the electron transport chain via ETF⅐ubiquinone oxidoreductase (3, 4). The acyl-CoA dehydrogenases, the first family of dehydrogenases identified to interact with ETF, are homotetramers with conserved tertiary and quaternary structures, one noncovalently bound FAD coenzyme molecule/protein subunit, and similar catalytic mechanisms (5-9). Very long chain, long chain, medium chain, and short chain (SCAD) acyl-CoA dehydrogenases catalyze the first step of fatty acid ␤-oxidation in the mitochondrial matrix. The other members of this family, including isovaleryl-CoA dehydrogenase (IVD), are involved in branched chain amino acid metabolism. The second family of dehydrogenases that interact with ETF consists of dimethylglycine dehydrogenase (DMGDH) and sarcosine dehydrogenase (SDH) (10 -12). These monomeric flavoproteins are active in choline catabolism. They also localize to the mitochondrial matrix but contain one molecule of covalently bound FAD/enz...

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“…To a solution of methylenecyclopentane (500 mg, 6.1 mmol) and 4-acetamidobenzaldehyde (1.1 g, 6.7 mmol) in 20 …”

Section: α-Cyclopent-1-en-1-yl-p-acetamidoacetophenone (2a)mentioning

confidence: 99%

Mechanism of an antibody-catalysed allylic isomerization

Gonçalves¹,

Dintinger²,

Lebreton

et al. 2000

Biochemical Journal

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The catalytic antibody 4B2, which was generated against a substituted amidine 1, catalyses the allylic isomerization of beta, gamma-unsaturated ketones with an acceleration factor (k(cat)/k(uncat)) of 1.5x10(3). On the basis of the 'bait and switch' strategy, it was reasoned that the positively charged hapten could elicit, by charge complementarity, an acidic residue (Asp or Glu) in the antibody-binding site in the right position to catalyse this proton transfer reaction. The pH dependence curve of k(cat)/K(m) shows a bell-shaped feature with an optimum at approx. pH 4.5. By cloning and sequencing the light and heavy chains of the 4B2 antibody, we confirmed the presence of several Asp and Glu residues in the complementarity-determining region loops. The antibody catalyses the alpha-proton exchange on the same substrates, demonstrating the involvement of a dienol intermediate in the reaction mechanism. Kinetic studies with (2)H-NMR provide evidence that alpha-proton abstraction is stereospecific. Whether the process involves one or two acid/base residues in this simple proton transfer or whether it is a concerted mechanism is discussed.

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3-Methylene-octanoyl-CoA and 3-methyl-trans-2-octenoyl-CoA: Two new mechanism-based inhibitors of medium chain acyl-CoA dehydrogenase from pig kidney

Cited by 24 publications

References 33 publications

A Novel Approach to the Characterization of Substrate Specificity in Short/Branched Chain Acyl-CoA Dehydrogenase

A Novel Approach to the Characterization of Substrate Specificity in Short/Branched Chain Acyl-CoA Dehydrogenase

Mammalian Electron Transferring Flavoprotein·Flavoprotein Dehydrogenase Complexes Observed by Microelectrospray Ionization-Mass Spectrometry and Surface Plasmon Resonance

Mechanism of an antibody-catalysed allylic isomerization

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