A method for resolution of general acyl-coenzyme A dehydrogenase apoprotein

Mayer, Ernest; Thorpe, Colin

doi:10.1016/0003-2697(81)90348-1

Cited by 44 publications

(42 citation statements)

References 3 publications

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“…The apoenzyme was prepared according to ref 35 and had a residual activity 1-2% that of the holoenzyme. To eliminate any residual activity, the apoenzyme was incubated for 30 min at 4°C with 5 equiv (compared with residual holoenzyme) 2-octynoyl-CoA (36).…”

Section: Methodsmentioning

confidence: 99%

Mechanism of Activation of Acyl-CoA Substrates by Medium Chain Acyl-CoA Dehydrogenase: Interaction of the Thioester Carbonyl with the Flavin Adenine Dinucleotide Ribityl Side Chain

et al. 1998

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The flavin adenine dinucleotide (FAD) cofactor of pig kidney medium-chain specific acylcoenzyme A (CoA) dehydrogenase (MCADH) has been replaced by ribityl-3′-deoxy-FAD and ribityl-2′-deoxy-FAD. 3′-Deoxy-FAD-MCADH has properties very similar to those of native MCADH, indicating that the FAD-ribityl side-chain 3′-OH group does not play any particular role in cofactor binding or catalysis. 2′-Deoxy-FAD-MCADH was characterized using the natural substrate C 8 CoA as well as various substrate and transition-state analogues. Substrate dehydrogenation in 2′-deoxy-FAD-MCADH is ≈1.5 × 10 7 -fold slower than that of native MCADH, indicating that disruption of the hydrogen bond between 2′-OH and substrate thioester carbonyl leads to a substantial transition-state destabilization equivalent to ≈38 kJ mol -1 . The RC-H microscopic pK a of the substrate analogue 3S-C 8 CoA, which undergoes R-deprotonation on binding to MCADH, is lowered from ≈16 in the free state to ≈11 ((0.5) when bound to 2′-deoxy-FAD-MCADH. This compares with a decrease of the same pK a to ≈5 in the complex with unmodified hwtMCADH, which corresponds to a pK shift of ≈11 pK units, i.e., ≈65 kJ mol -1 [Vock, P., Engst, S., Eder, M., and Ghisla, S. (1998) Biochemistry 37, 1848-1860]. The difference of this effect of ≈6 pK units (≈35 kJ mol -1 ) between MCADH and 2′-deoxy-FAD-MCADH is taken as the level of stabilization of the substrate carbanionic species caused by the interaction with the FAD-2′-OH. This energetic parameter derived from the kinetic experiments (stabilization of transition state) is in agreement with those obtained from static experiments (lowering of RC-H microscopic pK a of analogue, i.e., stabilization of anionic transition-state analogue). The contributions of the two single H-bonds involved in substrate activation (Glu376amide-N-H and ribityl-2′-OH) thus appear to behave additively toward the total effect. The crystal structures of native pMCADH and of 2′-deoxy-FAD-MCADH complexed with octanoyl-CoA/octenoyl-CoA show unambiguously that the FAD cofactor and the substrate/product bind in an identical fashion, implying that the observed effects are mainly due to (the absence of) the FAD-ribityl-2′-OH hydrogen bond. The large energy associated with the 2′-OH hydrogen bond interaction is interpreted as resulting from the changes in charge and the increased hydrophobicity induced by binding of lipophilic substrate. This is the first example demonstrating the direct involvement of a flavin cofactor side chain in catalysis.

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Section: Methodsmentioning

confidence: 99%

Mechanism of Activation of Acyl-CoA Substrates by Medium Chain Acyl-CoA Dehydrogenase: Interaction of the Thioester Carbonyl with the Flavin Adenine Dinucleotide Ribityl Side Chain

et al. 1998

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“…However, after exhaustive dialysis of the sampie against standard buffer, pH Determination of Phosphorus Content of pkMCADH Chemical analysis of phosphorus was performed according to the procedure described in Materials and Methods and revealed the presence of 3 ± 0.3 phosphorus atoms per subunit of pkMCADH. Since the protein has tigthly bo- Mayer and Thorpe, 1981) two phosphorus atoms are due to the FAD content. In contrast to the pig kidney enzyrne, analysis of recombinant Y375G-MCADH expressed in and isolated from E. coli showed the presence of 2 ± 0.35 phosphorus atoms per enzyme subunit.…”

Section: P-nmr Studiesmentioning

confidence: 99%

“…In contrast, dephosphorylated pkMCADH shows a decreased solubility (0.2 -0.3 mM under the Sal11e conditions). When preparations of apo-pkMCADH are carried out according to the method of Mayer and Thorpe (1981) high yields (> 75%) are obtained when starting with native enzyme. However, with dephosphorylated pkMCADH the yields were < 10%.…”

Section: Influence Of the Phosphomonoester On The Solubility And Apo-mentioning

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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“…The presence of green 6-hydroxy-FAD [7], which could have been responsible for the green colour of the reductase, was excluded. When the FAD was removed from the enzyme under milder conditions, treatment with ammonium sulfate and KBr at pH 2 in the presence of charcoal [8], a colourless inactive protein was obtained which could be reactivated by FAD, but not by FMN, to 200% of the original activity. After removal of the excess FAD by repeated washings through an ultrafiltration membrane, a yellow active enzyme with a normal flavoprotein spectrum was obtained.…”

Section: Molecular Propertiesmentioning

confidence: 99%

A green 2,4‐pentadienoyl‐CoA reductase from Clostridium aminovalericum

Eikmanns

1991

European Journal of Biochemistry

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2,CPentadienoyl-CoA reductase from Clostridium aminovalericum was purified to homogeneity (170 -182 kDa). PAGE in the presence of SDS revealed a single band (44 kDa) indicating a homotetrameric structure. The native enzyme had a green colour and contained 0.4 mol FAD/subunit. Its unusual ultraviolet/visiblespectrum showed absorption maxima at 270,402 and 715 nm as well as shoulders at 278, 360,450 and 500 nm. Removal of the prosthetic group at pH 2 in the presence of salt and charcoal yielded a colourless and completely inactive apoenzyme, which could be reconstituted with FAD (not with FMN) to an active holoenzyme showing a normal flavoprotein spectrum (peaks at 369 nm and 436 nm). Thereby the FAD content increased to 0.9 mol/ subunit with a concomitant rise in activity to 200% of the original value. Anaerobic reduction of the green enzyme by dithionite and reoxidation by air afforded a green preparation with a spectrum similar to that of the native enzyme. Addition of excess FAD to the green reductase also increased the activity by a factor of two.The green enzyme catalysed the oxidation of (E)-3-pentenoyl-CoA or (E)-3-hexenoyl-CoA to 2,4-pentadienoylCoA or 2,4-hexenoyl-CoA, respectively. 2-Pentenoyl-CoA or 4-pentenoyl-CoA were not oxidised. Meldola blue (8-dimethylamino-2,3-benzophenoxazine) and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride ( V = 26 nkat/mg protein) or ferricenium hexafluorophosphate ( V = 1900 nkat/mg), but not NAD(P), served as electron acceptors. Reduction of 2,4-pentadienoyl-CoA ( V = 370 nkat/mg) was observed with reduced benzyl viologen, but not with NAD(P)H as an electron donor. Although the enzyme had some pentenoyl-CoA A-isomerase activity (1.2 nkat/mg), the only product of the reduction was 3-pentenoyl-CoA rather than 2-pentenoylCoA.

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A method for resolution of general acyl-coenzyme A dehydrogenase apoprotein

Cited by 44 publications

References 3 publications

Mechanism of Activation of Acyl-CoA Substrates by Medium Chain Acyl-CoA Dehydrogenase: Interaction of the Thioester Carbonyl with the Flavin Adenine Dinucleotide Ribityl Side Chain

Mechanism of Activation of Acyl-CoA Substrates by Medium Chain Acyl-CoA Dehydrogenase: Interaction of the Thioester Carbonyl with the Flavin Adenine Dinucleotide Ribityl Side Chain

Medium-Chain Acyl CoA Dehydrogenase: Evidence for Phosphorylation

A green 2,4‐pentadienoyl‐CoA reductase from Clostridium aminovalericum

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