Purification and properties of electron-transferring flavoprotein from pig kidney

Gorelick, Robert J.; Mizzer, John P.; Thorpe, Colin

doi:10.1021/bi00269a049

Cited by 68 publications

(71 citation statements)

References 35 publications

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“…Hydratase activity was measured by following the decrease in absorbance at 280 nm observed upon hydration of the a,P double bond (Steinman & Hill, 1975;see Methods). The dehydrogenase was purified as described earlier (Gorelick et al, 1982), except that the Cibacron Blue-Sepharose column was replaced by a gel filtration step (Sephacryl S-200, Table I; Thorpe et al, 1979). Various batches of enzyme prepared in this way showed considerable variation in hydratase activity while exhibiting essentially the same turnover number in the standard acyl-CoA dehydrogenase assay (190 m i d ; Thorpe et al, 1979).…”

Section: Resultsmentioning

confidence: 99%

Medium-chain acyl coenzyme A dehydrogenase from pig kidney has intrinsic enoyl coenzyme A hydratase activity

Lau¹,

Powell²,

Buettner³

et al. 1986

Biochemistry

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ABSTRACT:The flavoprotein medium-chain acyl coenzyme A (acyl-CoA) dehydrogenase from pig kidney exhibits an intrinsic hydratase activity toward crotonyl-CoA yielding L-3-hydroxybutyryl-CoA. The maximal turnover number of about 0.5 min-' is 500-1000-fold slower than the dehydrogenation of butyryl-CoA using electron-transferring flavoprotein as terminal acceptor. trans-2-Octenoyl-and trans-2-hexadecenoyl-CoA are not hydrated significantly. Hydration is not due to contamination with the short-chain enoyl-CoA hydratase crotonase. Several lines of evidence suggest that hydration and dehydrogenation reactions probably utilize the same active site. These two activities are coordinately inhibited by 2-octynoyl-CoA and (methylenecyclopropy1)acetyLCoA [whose targets are the protein and flavin adenine dinucleotide (FAD) moieties of the dehydrogenase, respectively]. The hydration of crotonyl-CoA is severely inhibited by octanoyl-CoA, a good substrate of the dehydrogenase. The apoenzyme is inactive as a hydratase but recovers activity on the addition of FAD. Compared with the hydratase activity of the native enzyme, the 8-fluoro-FAD enzyme exhibits a roughly 2-fold increased activity, whereas the 5-deaza-FAD dehydrogenase is only 20% as active.

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Section: Resultsmentioning

confidence: 99%

Medium-chain acyl coenzyme A dehydrogenase from pig kidney has intrinsic enoyl coenzyme A hydratase activity

Lau¹,

Powell²,

Buettner³

et al. 1986

Biochemistry

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“…The potential of the semiquinone-dihydroquinone couple is more conventional (-197 mV; (17)), indicating that there is a substan-tial (and essentially complete) kinetic block on full reduction of ETF by dithionite (-530 mV) or photoexcited deazariboflavin (-650 mV). A similar, albeit less complete, kinetic block on reduction to the dihydroquinone has been reported for pig liver ETF; reduction to the dihydroquinone level in pig liver ETF requires about 1 h for equilibration (13,14). The potentials measured for free M. methylotrophus ETF (as with any ETF) may not of course reflect the situation in the electron transfer complex with its physiological electron donor (TMADH) but nevertheless are likely to serve as a reasonable guide.…”

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confidence: 80%

“…The ETF from M. elsdenii is unusual in acting physiologically as a two-electron carrier. ETF from mammalian sources and P. denitrificans can be reduced to the dihydroquinone form, by reduction with dithionite or by photoreduction (13)(14)(15), although reduction to the two-electron level is relatively slow. M. methylotrophus ETF is readily converted to the semiquinone form in reactions with its physiological electron donor, trimethylamine dehydrogenase (TMADH) (4), or during artificial reduction with dithionite (16).…”

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confidence: 99%

αArg-237 in Methylophilus methylotrophus (sp. W3A1) Electron-transferring Flavoprotein Affords ∼200-Millivolt Stabilization of the FAD Anionic Semiquinone and a Kinetic Block on Full Reduction to the Dihydroquinone

Talfournier¹,

Munro

Basran³

et al. 2001

Journal of Biological Chemistry

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The midpoint reduction potentials of the FAD cofactor in wild-type Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein (ETF) and the ␣R237A mutant were determined by anaerobic redox titration. The FAD reduction potential of the oxidizedsemiquinone couple in wild-type ETF (E 1 ) is ؉153 ؎ 2 mV, indicating exceptional stabilization of the flavin anionic semiquinone species. Conversion to the dihydroquinone is incomplete (E 2 < ؊250 mV), because of the presence of both kinetic and thermodynamic blocks on full reduction of the FAD. A structural model of ETF (Chohan, K. K., Scrutton, N. S., and Sutcliffe, M. J. (1998) Protein Pept. Lett. 5, 231-236) suggests that the guanidinium group of Arg-237, which is located over the si face of the flavin isoalloxazine ring, plays a key role in the exceptional stabilization of the anionic semiquinone in wild-type ETF. The major effect of exchanging ␣Arg-237 for Ala in M. methylotrophus ETF is to engineer a remarkable ϳ200-mV destabilization of the flavin anionic semiquinone (E 2 ‫؍‬ ؊31 ؎ 2 mV, and E 1 ‫؍‬ ؊43 ؎ 2 mV). In addition, reduction to the FAD dihydroquinone in ␣R237A ETF is relatively facile, indicating that the kinetic block seen in wild-type ETF is substantially removed in the ␣R237A ETF. Thus, kinetic (as well as thermodynamic) considerations are important in populating the redox forms of the protein-bound flavin. Additionally, we show that electron transfer from trimethylamine dehydrogenase to ␣R237A ETF is severely compromised, because of impaired assembly of the electron transfer complex.

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“…Human wild type MCADH was obtained as described elsewhere (Kieweg et al, 1997). ETF was purified as described by Gorelick et al (1982).…”

Section: Chemieals and Enzymesmentioning

confidence: 99%

Medium-Chain Acyl CoA Dehydrogenase: Evidence for Phosphorylation

Macheroux¹,

Sanner²,

Büttner³

et al. 1997

Biological Chemistry

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Mature medium chain acyl-CoA dehydrogenase isolated from pig kidney (pkMCADH) and originating from mitochondria carries a phosphate group as demonstrated by 31 P-NMR-spectroscopy and chemical analysis. Two broad resonances at -6.3 and -8 ppm are observed and are assigned to the pyrophosphate group of the cofactor FAD. A third, narrow resonance at 4.65 ppm indicates the presence of a phosphomonoester residue. Chemical analysis of intact pkMCADH shows the presence of 3 ± 0.3 phosphates, those of FAD and of an additional covalently attached phosphate. With recombinant, human wild type MCADH expressed in and purified from E. coli only the two FAD phosphates (2 ± 0.35) are found. Similarly, pkMCADH which has been converted to the apoenzyme and reconstituted to holoenzyme also contains 2 ± 0.4 phosphates. The covalently bound phosphate can be hydrolyzed by phosphatase and subsequently removed by dialysis. The phosphate group has no detectable effect on the catalytic activity of the MCADH measured with artificial and natural electron acceptors such as pig electron transferring flavoprotein. However, phosphorylation has a marked effect on protein solubility which is ffi5-fold lower for the dephosphorylated protein.

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Purification and properties of electron-transferring flavoprotein from pig kidney

Cited by 68 publications

References 35 publications

Medium-chain acyl coenzyme A dehydrogenase from pig kidney has intrinsic enoyl coenzyme A hydratase activity

Medium-chain acyl coenzyme A dehydrogenase from pig kidney has intrinsic enoyl coenzyme A hydratase activity

αArg-237 in Methylophilus methylotrophus (sp. W3A1) Electron-transferring Flavoprotein Affords ∼200-Millivolt Stabilization of the FAD Anionic Semiquinone and a Kinetic Block on Full Reduction to the Dihydroquinone

Medium-Chain Acyl CoA Dehydrogenase: Evidence for Phosphorylation

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