Inhibition of general acyl CoA dehydrogenase by electron transfer flavoprotein semiquinone

Beckmann, Joe D.; Frerman, Frank E.; McKean, Martha C.

doi:10.1016/s0006-291x(81)80151-9

Cited by 45 publications

(21 citation statements)

References 9 publications

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“…In this investigation, we have demonstrated for the first time reverse electron flow from purified ETF-QO to ETF. Furthermore, submitochondrial particles exhibit NADH-ETF oxidoreductase activity that is completely inhibited by antibodies directed against ETF-Q0.3 Since ETF semiquinone is known to be a potent product inhibitor of G-AD (Beckmann et al, 1981), it is reasonable to suggest that the rate of P-oxidation will be governed by the mitochondrial redox B E C K M A N N A N D F R E R M A N state. The ratio of oxidized to reduced ETF will be dependent upon the NADH/NAD ratio.…”

Section: Discussionmentioning

confidence: 99%

Electron transfer flavoprotein-ubiquinone oxidoreductase from pig liver: purification and molecular, redox, and catalytic properties

Beckmann¹,

Frerman²

1985

Biochemistry

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Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) was purified to homogeneity from pig liver submitochondrial particles. It is comparable in molecular weight and general properties to ETF-QO from beef heart [Ruzicka, F. J., & Beinert, H. (1977) J. Biol. Chem. 252, 8440-8445], and the electron spin resonance signals of the reduced iron-sulfur cluster are essentially identical. ETF-QO catalyzes the transfer of electrons from electron-transfer flavoprotein (ETF) to nitro blue tetrazolium, with a sluggish reaction turnover number of about 10-30 min-1. In contrast, the enzyme rapidly disproportionates ETF semiquinone, with a turnover number of 200 s-1. The reverse reaction, comproportionation of oxidized and hydroquinone ETF, provides an enzymatic assay for ETF-QO with picomolar sensitivity. Equilibrium spectrophotometric titrations show that ETF-QO accepts a maximum of two electrons from ETF and accepts three electron equivalents from dithionite or by photochemical reduction. All electrons from the enzymatically or chemically reduced protein can be transferred to 2,3-dimethoxy-5-methyl-6-pentyl-1,4-benzoquinone (PB), and this reaction is readily reversible. Reduction of ETF-QO by 2,3-dimethoxy-5-methyl-6-pentyl-1,4-benzohydroquinone is pH dependent and indicates the enzyme to have a redox potential that decreases by 47 mV per pH unit. Therefore, ETF-QO binds one to two protons upon reduction. The EO' at pH 7.3 is 38 mV. The ability of ETF-QO to catalyze the equilibration of ETF redox states has been used to evaluate the equilibrium 2ETFsq + nH+ in equilibrium ETFox + ETFhq.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionmentioning

confidence: 99%

Electron transfer flavoprotein-ubiquinone oxidoreductase from pig liver: purification and molecular, redox, and catalytic properties

Beckmann¹,

Frerman²

1985

Biochemistry

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“…The DCIP reduction assay was performed as described (Hall, 1978;Mohsen & Vockley, 1995). The ETF reduction assay was performed using an LS50B fluorescence spectrophotometer from Perkin Elmer (Norwalk, CT) with a heated cuvette block set to 32°C as described (Beckmann et al, 1981;Mohsen & Vockley, 1995). For kinetic analysis, ETF concentration was 1 µM and substrate concentration was varied from 1 to 50 µM final concentration and k m and V max were calculated on a Macintosh computer using the EnzymeKinetics V 1.4.1 software from Trinity Software (Campton, NH).…”

Section: Growth Of Uracil-containing Phagemids For In Vitromentioning

confidence: 99%

Functional Role of the Active Site Glutamate-368 in Rat Short Chain Acyl-CoA Dehydrogenase

1996

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The acyl-CoA dehydrogenases are a family of flavoenzymes with similar structure and function involved in the metabolism of fatty acids and branched chain amino acids. The degree of overlap in substrate specificity is narrow among these enzymes. The position of the catalytic glutamate, identified as Glu376 in porcine medium chain acyl-CoA dehydrogenase (MCAD), Glu254 in human isovaleryl-CoA dehydrogenase (IVD), and Glu261 in human long chain acyl-CoA dehydrogenase (LCAD), has been suggested to affect substrate chain length specificity. In this study, in vitro site-directed mutagenesis was used to investigate the effect of changing the position of the catalytic carboxylate on substrate specificity in short chain acyl-CoA dehydrogenase (SCAD). Glu368, the hypothetical active site catalytic residue of rat SCAD, was replaced with Asp, Gly, Gln, Arg, and Lys and the wild type and mutant SCADs were produced in Escherichia coli and purified. The recombinant wild type SCAD kcat/K(m) values for butyryl-hexanoyl-, and octanoyl-CoA were 220, 22, and 3.2 microM-1 min-1, respectively, while the Glu368Asp mutant gave kcat/K(m) of 81, 12, and 1.4 microM-1 min-1, respectively, for the same substrates. None of the other mutants exhibited enzyme activity. A Glu368Gly/Gly247Glu double mutant enzyme, which places the catalytic residue at a position homologous to that of LCAD, was also synthesized and purified. It showed kcat/K(m) of 9.3, 2.8, and 1.5 microM-1 min-1 with butyryl-, hexanoyl-, and octanoyl-CoA used as substrates, respectively. These results confirm the identity of Glu368 as the catalytic residue of rat SCAD and suggest that alteration of the position of the catalytic carboxylate can modify substrate specificity.

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“…Similarly deficiency of ETF or ETF: QO leads to diminished P-oxidation flux and an accumulation of long-chain saturated acyl-CoA and and acylcarnitine esters (Singh Kler et al, 1991). ETF-semiquinone, the partially reduced form of ETF, can be produced when the coenzyme Q pool is excessively reduced (Frerman, 1987) and is a potent inhibitor of the acyl-CoA dehydrogenase (Beckmann et al, 1981), so that the activity of the acyl-CoA dehydrogenases may be.responsive to the redox state of the coenzyme Q pool. Little work has been carried out on the importance of this inhibition in the intact mitochondrion.…”

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confidence: 99%

Redox control of β‐oxidation in rat liver mitochondria

Eaton

Turnbull

Bartlett

1994

European Journal of Biochemistry

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Coupled rat liver mitochondria were incubated with [U-i4C]hexadecanoate and carnitine which resulted in the formation of acyl-, 2-enoyl-and 3-hydroxyacyl-CoA and carnitine esters. The production of 2-enoyl-CoA and 3-hydroxyacyl-CoA esters was associated with a significant lowering of the NAD'NADH ratio, in contrast to rat muscle mitochondria [Eaton, S., Bhuiyan, A. K. M. J., Kler, R. S., Turnbull, D. M. & Bartlett, K. (1993) Biochem. J. 289, 161-1721, suggesting that control by the respiratory chain is important under normal conditions. When NAD'NADH ratios were held low by succinate-induced reverse electron flow, 3-enoyl-CoA esters were also detected, probably formed by the action of 3,2-enoyl-CoA isomerase. Measurement of the flux of P-oxidation at different osmolalities showed that flux was strongly dependent on osmolality changes in the physiological range. Measurement of the CoA and carnitine esters resulting from incubations made at different osmolalities showed that there was an increase in the amounts of the saturated acyl-CoA esters with respect to 2-enoyl-CoA and 3-hydroxyacyl-CoA esters, consistent with control by the electron-transfer flavoprotein-ubiquinone segment [Halestrap, A. P. & Dunlop, J. L. (1986) Biochem. J. 239, 559-5651. This however could not be the only factor operating as indicated by the continued presence of 2-enoyl-CoA and 3-hydroxyacyl-CoA esters at high osmolalities.Mitochondria1 P-oxidation is linked to the respiratory chain at two stages ; the 3-hydroxyacyl-CoA dehydrogenases to complex I via NAD'DJADH and the acyl-CoA dehydrogenases to ubiquinone via electron-transfer flavoprotein (ETF) and its oxidoreductase (ETF: QO). Inhibition of respiratory-chain activity at the level of complex I leads to diminished P-oxidation flux and the accumulation of 3-hydroxyacyl-CoA and 2-enoyl-CoA and carnitine esters (Bremer and

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Inhibition of general acyl CoA dehydrogenase by electron transfer flavoprotein semiquinone

Cited by 45 publications

References 9 publications

Electron transfer flavoprotein-ubiquinone oxidoreductase from pig liver: purification and molecular, redox, and catalytic properties

Electron transfer flavoprotein-ubiquinone oxidoreductase from pig liver: purification and molecular, redox, and catalytic properties

Functional Role of the Active Site Glutamate-368 in Rat Short Chain Acyl-CoA Dehydrogenase

Redox control of β‐oxidation in rat liver mitochondria

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