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Colin Thorpe - One of the best experts on this subject based on the ideXlab platform.
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4-Hydroxycinnamoyl-CoA: an ionizable probe of the active site of the medium chain Acyl-CoA Dehydrogenase.
Biochemistry, 2000Co-Authors: Irina Rudik, Alasdair F. Bell, Peter J. Tonge, Colin ThorpeAbstract:4-OH-Cinnamoyl-CoA has been synthesized as a probe of the active site in the medium chain Acyl-CoA Dehydrogenase. The protonated form of the free ligand (λmax = 336 nm) yields the corresponding phenolate (λmax = 388 nm) with a pK of 8.9. 4-OH-Cinnamoyl-CoA binds tightly (Kd = 47 nM, pH 6) to the pig kidney Dehydrogenase with a prominent new band at 388 nm, suggesting ionization of the bound ligand. However, this spectrum reflects polarization, not deprotonation, of the neutral form of the ligand. Thus, the 388 nm band is abolished as the pH is raised (not lowered), and analogous spectral and pH behavior is observed with the nonionizable analogue 4-methoxycinnamoyl-CoA. Studies with wild type, E99G, and E376Q mutants of the human medium chain Acyl-CoA Dehydrogenase showed that these two active site carboxylates strongly suppress ionization of the 4-OH ligand. Binding to the double mutant E99G/E376Q gives an intense new band as the pH is raised (pK = 7.8), with an absorbance maximum at 498 nm resembling the...
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Protonic Equilibria in the Reductive Half-Reaction of the Medium-Chain Acyl-CoA Dehydrogenase †
Biochemistry, 1998Co-Authors: Irina Rudik, Sandro Ghisla, Colin ThorpeAbstract:Oxidation of thioester substrates in the Medium-Chain Acyl-CoA Dehydrogenase involves α-proton abstraction by the catalytic base, Glu376, with transfer of a β-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....
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Medium-Chain Acyl-CoA Dehydrogenase- and enoyl-CoA hydratase-dependent bioactivation of 5,6-dichloro-4-thia-5-hexenoyl-CoA.
Biochemistry, 1995Co-Authors: Michael E. Fitzsimmons, Colin Thorpe, M. W. AndersAbstract: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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Reductive half-reaction in Medium-Chain Acyl-CoA Dehydrogenase: modulation of internal equilibrium by carboxymethylation of a specific methionine residue.
Biochemistry, 1992Co-Authors: John G. Cummings, Sze Mei Lau, Patricia J. Powell, Colin ThorpeAbstract:Pig kidney Medium-Chain Acyl-CoA Dehydrogenase is specifically alkylated at a methionine residue by treatment with iodoacetate at pH 6.6. This residue corresponds to Met249 in the human Medium-Chain Acyl-CoA Dehydrogenase sequence [Kelly, D. P., Kim, J. J., Billadello, J. J., Hainline, B. E., Chu, T. W., & Strauss, A. W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4068-4072]. The S-carboxymethylated Dehydrogenase shows a drastically lowered affinity for octanoyl-CoA (from submicromolar to 65 microM), but retains about 23% of the maximal activity of the native enzyme. In addition, alkylation perturbs the internal redox equilibrium: E.FADox.octanoyl-CoA K2 in equilibrium with E.FAD2e.octenoyl-CoA K2 ranges from about 9 for the native enzyme to about 0.2 for the homogeneously modified protein. This effect is not due to a significant change in the redox potential of the free enzyme upon alkylation. Rather, carboxymethylation weakens the preferential binding of enoyl-CoA product to the reduced enzyme (K3) compared to octanoyl-CoA binding to the oxidized Dehydrogenase (K1) that is required to pull the substrate thermodynamically uphill. Thus, the ratio of dissociation constants, K1/K3, decreases from about 15,000 for the native enzyme to only 330 upon carboxymethylation of Met249. Binding studies with a variety of Acyl-CoA analogues and manipulation of enzyme redox potentials by substitution of the natural prosthetic group by 8-Cl-FAD confirm the thermodynamic effects of alkylation.
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Reactivity of Medium-Chain Acyl-CoA Dehydrogenase toward molecular oxygen.
Biochemistry, 1991Co-Authors: Rong Wang, Colin ThorpeAbstract:The free two-electron-reduced form of Medium-Chain Acyl-CoA Dehydrogenase is reoxidized by 120 microM molecular oxygen (50 mM phosphate buffer, pH 7.6, 2 degrees C) with a half-time of approximately 7 s. Reoxidation yields hydrogen peroxide as a major product with only traces of the superoxide anion. In contrast, enzyme reduced with octanoyl-CoA is extremely slowly reoxidized oxygen, and so a series of 14 different substrate analogues have been tested to assess the structural factors responsible for this effect. Complexes with redox-inactive ligands such as 3-thia- and 2-azaoctanoyl-CoA lead to an approximately 3000-fold slowing of the rate of reoxidation of the free dihydroflavin form of the enzyme. Comparable ligands lacking the thioester carbonyl function are much less effective with rates some 1.3-4-fold slower than the free enzyme. The strong suppression of oxygen reactivity observed with certain ligands is probably not simply a steric effect but may reflect desolvation of the active site and consequent destabilization of the superoxide anion intermediate formed during reoxidation of the flavin. The profound differences in oxygen reactivity between Acyl-CoA Dehydrogenase and Acyl-CoA oxidase and the unusual stability of certain flavoprotein semiquinones in air are discussed in terms of these thermodynamic and kinetic arguments.
Muin J Khoury - One of the best experts on this subject based on the ideXlab platform.
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The epidemiology of medium chain Acyl-CoA Dehydrogenase deficiency: An update
Genetics in Medicine, 2006Co-Authors: Scott D Grosse, Krista S Crider, Carol L Greene, Muin J Khoury, Rodney J PollittAbstract:The most common fatty acid oxidation disorder, medium chain Acyl-CoA Dehydrogenase deficiency (MCADD), has become the focal point for the adoption of tandem mass spectrometry to detect it and related inborn errors of metabolism. This article updates a human genome epidemiology review of MCADD published in 1999. The focus of this update is on epidemiologic parameters rather than mutations associated with MCADD. Currently available information from screening studies on the frequency of detection of MCADD in newborns, as well as the frequency of homozygotes for the common mutation in the ACADM gene, is summarized. In the United States, the average incidence of the disorder is from 1 in 15,000 to 1 in 20,000 births, with individual states reporting frequencies from 1 in 10,000 to 1 in 30,000 births. In addition, a systematic review was undertaken of the published literature on the frequency of mortality and developmental disabilities among children with MCADD, both in screened and unscreened cohorts. It seems that in the absence of newborn screening for MCADD, premature death or serious disability occurs in 20% to 25% of children with the disorder. Systematic collection and analysis of follow-up data are still needed to ascertain the frequencies of outcomes in screened cohorts.
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Is the G985A allelic variant of Medium-Chain Acyl-CoA Dehydrogenase a risk factor for sudden infant death syndrome? A pooled analysis.
Pediatrics, 2000Co-Authors: Sophia S. Wang, Paul M. Fernhoff, Muin J KhouryAbstract:To the Editor. Studies examining the relationship between Medium-Chain Acyl-CoA Dehydrogenase deficiency (MCADD) and sudden infant death syndrome (SIDS) have shown conflicting results. With >90% of individuals diagnosed with MCADD possessing at least one copy of the G985A allelic variant, it seems likely that if an association between MCADD and SIDS existed, an association would also be seen between the G985A and SIDS. …
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An epidemiologic assessment of the relationship between the G985A medium chain Acyl-CoA Dehydrogenase deficiency (MCADD) allelic variant and sudden infant death syndrome (SIDS)
Genetics in Medicine, 1999Co-Authors: Sophia S. Wang, Muin J KhouryAbstract:An epidemiologic assessment of the relationship between the G985A medium chain Acyl-CoA Dehydrogenase deficiency (MCADD) allelic variant and sudden infant death syndrome (SIDS)
J V Leonard - One of the best experts on this subject based on the ideXlab platform.
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Prospective surveillance study of medium chain Acyl-CoA Dehydrogenase deficiency in the UK
Archives of disease in childhood, 1998Co-Authors: Rodney J Pollitt, J V LeonardAbstract:BACKGROUND—Medium chain Acyl-CoA Dehydrogenase (MCAD) deficiency is a common disorder of fatty acid oxidation in north west Europe. It is very variable in its clinical consequences and is believed to be considerably underdiagnosed. OBJECTIVE—To investigate the diagnosis and outcome of MCAD deficiency in the UK. METHOD—A prospective surveillance study through the British Paediatric Surveillance Unit. RESULTS—Of 62 affected individuals identified, 57 were from England, giving an incidence of 4.5 cases/100 000 births. Forty six cases presented with an acute illness (10 of whom died), 13 cases were identified because of family history, and three for other reasons. Six of the survivors were neurologically impaired. CONCLUSIONS—Despite increased clinical awareness, the mortality and morbidity from MCAD deficiency remain high. The frequency and severity of the disease support the case for the introduction of universal neonatal screening in England and Scotland.
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Rapid diagnosis of Medium-Chain acyl CoA Dehydrogenase deficiency by measurement ofcis-4-decenoic acid in plasma
Journal of Inherited Metabolic Disease, 1991Co-Authors: Simon J. R. Heales, D. A. Woolf, P Robinson, J V LeonardAbstract:Plasma concentrations of octanoate and cis -4-decenoate were measured by gas chromatography-mass spectrometry in children with deficiencies of Medium-Chain Acyl-CoA Dehydrogenase (MCAD), long-chain 3-hydroxyAcyl-CoA Dehydrogenase (3LHAD) and multiple Acyl-CoA Dehydrogenase (MAD) deficiency. Children receiving medium- and long-chain lipid supplements were also studied. Octanoate was elevated in all but one of the children with MCAD deficiency, in MAD deficiency and in children receiving Medium-Chain triglyceride supplementation. Cis -4-decenoate was only elevated in MCAD and MAD deficiency. It is concluded that measurement of plasma cis -4-decenoate provides a sensitive and specific test for defects of Medium-Chain acyl CoA Dehydrogenase.
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Fatty infiltration in the liver in medium chain acyl CoA Dehydrogenase deficiency.
Archives of disease in childhood, 1991Co-Authors: H C Losty, P Lee, M Alfaham, O P Gray, J V LeonardAbstract:Fatty infiltration of the liver at postmortem examination has been recommended as a criterion for selection of infants who have died suddenly and unexpectedly for further biochemical investigation for disorders of fatty acid oxidation. We describe a boy with medium chain acyl CoA Dehydrogenase deficiency who died four months after diagnosis and in whom only minimal hepatic fatty infiltration was found.
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Rapid diagnosis of Medium-Chain acyl CoA Dehydrogenase deficiency by measurement of cis -4-decenoic acid in plasma
Journal of inherited metabolic disease, 1991Co-Authors: Simon J. R. Heales, D. A. Woolf, P Robinson, J V LeonardAbstract:Plasma concentrations of octanoate andcis-4-decenoate were measured by gas chromatography-mass spectrometry in children with deficiencies of Medium-Chain Acyl-CoA Dehydrogenase (MCAD), long-chain 3-hydroxyAcyl-CoA Dehydrogenase (3LHAD) and multiple Acyl-CoA Dehydrogenase (MAD) deficiency. Children receiving medium- and long-chain lipid supplements were also studied. Octanoate was elevated in all but one of the children with MCAD deficiency, in MAD deficiency and in children receiving Medium-Chain triglyceride supplementation.Cis-4-decenoate was only elevated in MCAD and MAD deficiency. It is concluded that measurement of plasmacis-4-decenoate provides a sensitive and specific test for defects of Medium-Chain acyl CoA Dehydrogenase.
Sophia S. Wang - One of the best experts on this subject based on the ideXlab platform.
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Is the G985A allelic variant of Medium-Chain Acyl-CoA Dehydrogenase a risk factor for sudden infant death syndrome? A pooled analysis.
Pediatrics, 2000Co-Authors: Sophia S. Wang, Paul M. Fernhoff, Muin J KhouryAbstract:To the Editor. Studies examining the relationship between Medium-Chain Acyl-CoA Dehydrogenase deficiency (MCADD) and sudden infant death syndrome (SIDS) have shown conflicting results. With >90% of individuals diagnosed with MCADD possessing at least one copy of the G985A allelic variant, it seems likely that if an association between MCADD and SIDS existed, an association would also be seen between the G985A and SIDS. …
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An epidemiologic assessment of the relationship between the G985A medium chain Acyl-CoA Dehydrogenase deficiency (MCADD) allelic variant and sudden infant death syndrome (SIDS)
Genetics in Medicine, 1999Co-Authors: Sophia S. Wang, Muin J KhouryAbstract:An epidemiologic assessment of the relationship between the G985A medium chain Acyl-CoA Dehydrogenase deficiency (MCADD) allelic variant and sudden infant death syndrome (SIDS)
Irina Rudik - One of the best experts on this subject based on the ideXlab platform.
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4-Hydroxycinnamoyl-CoA: an ionizable probe of the active site of the medium chain Acyl-CoA Dehydrogenase.
Biochemistry, 2000Co-Authors: Irina Rudik, Alasdair F. Bell, Peter J. Tonge, Colin ThorpeAbstract:4-OH-Cinnamoyl-CoA has been synthesized as a probe of the active site in the medium chain Acyl-CoA Dehydrogenase. The protonated form of the free ligand (λmax = 336 nm) yields the corresponding phenolate (λmax = 388 nm) with a pK of 8.9. 4-OH-Cinnamoyl-CoA binds tightly (Kd = 47 nM, pH 6) to the pig kidney Dehydrogenase with a prominent new band at 388 nm, suggesting ionization of the bound ligand. However, this spectrum reflects polarization, not deprotonation, of the neutral form of the ligand. Thus, the 388 nm band is abolished as the pH is raised (not lowered), and analogous spectral and pH behavior is observed with the nonionizable analogue 4-methoxycinnamoyl-CoA. Studies with wild type, E99G, and E376Q mutants of the human medium chain Acyl-CoA Dehydrogenase showed that these two active site carboxylates strongly suppress ionization of the 4-OH ligand. Binding to the double mutant E99G/E376Q gives an intense new band as the pH is raised (pK = 7.8), with an absorbance maximum at 498 nm resembling the...
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Protonic Equilibria in the Reductive Half-Reaction of the Medium-Chain Acyl-CoA Dehydrogenase †
Biochemistry, 1998Co-Authors: Irina Rudik, Sandro Ghisla, Colin ThorpeAbstract:Oxidation of thioester substrates in the Medium-Chain Acyl-CoA Dehydrogenase involves α-proton abstraction by the catalytic base, Glu376, with transfer of a β-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....