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

Lau, Sze Mei; Powell, Pat; Buettner, Hermann; Ghisla, Sandro; Thorpe, Colin

doi:10.1021/bi00363a003

Cited by 45 publications

(43 citation statements)

References 45 publications

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“…The interpretation of Reinsch et al (irreversible reaction) [6] might have relied on their supposedly observed lack of reaction of reduced enzyme with crotonyl-CoA. It is conceivable that their results [6] have been affected by the relative instability of this unsaturated substrate, especially in view of our findings that crotonase (hydratase) activities can be present in acyl-CoA dehydrogenase preparations [26].…”

Section: Discussionmentioning

confidence: 68%

“…Steps k7 -will occur subsequently to anaerobic reduction, they represent dissociation of product and attainment of a thermodynamic equilibrium involving all species shown. This equilibration is likely to be connected to the third slow kinetic phase; it will, however, be more complicated than shown since product is also transformed, by the endogenous hydratase activity described elsewhere [26], to b-hydroxy-butyryl-CoA and since the latter also will bind to oxidized and reduced GAD.…”

Section: Discussionmentioning

confidence: 96%

“…[S]) and might involve a (slow) endogenous hydratase activity of GAD as detailed elsewhere [26]. The extent of reduction observed during the three phases is strongly dependent on the substrate concentration, as will be detailed below (cf.…”

Section: Steady-state Kinetics Using Undeuterated Selectively Deutermentioning

confidence: 97%

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Studies on the reaction mechanism of general acyl‐CoA dehydrogenase

Pohl

Raichle

Ghisla

1986

European Journal of Biochemistry

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The kinetic properties of general acyl‐CoA dehydrogenase from pig kidney have been investigated using normal butyryl‐CoA as well as a α‐deutero, β‐deutero‐and perdeutero‐butyryl‐CoA. In turnover catalysis, isotope effects of 2, 3.6, and 9 were found respectively. In the reductive half reaction the isotope effects were 2.5, 14, and 28 for the same substrates, and 21 for (2R,3R)‐(2,3‐D2)butyryl‐CoA. No intermediates are apparent during the reduction of oxidized enzyme to the presumed complex of reduced enzyme and crotonyl‐CoA. The results are interpreted as indicating a high degree of concertedness during the rupture of the α and β C‐H bonds. They are compatible with a mechanism in which simultaneously the α‐hydrogen is abstracted as a proton, while the β‐hydrogen is transferred to the oxidized flavin as a hydride.

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Section: Discussionmentioning

confidence: 68%

Section: Discussionmentioning

confidence: 96%

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Studies on the reaction mechanism of general acyl‐CoA dehydrogenase

Pohl

Raichle

Ghisla

1986

European Journal of Biochemistry

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“…1-amino-2-naphtol-4-sulfonic acid was from Merck, alkaline phosphatase, PMS from Sigma and DCI frorn Fluka, ferricenium hexafluorophosphate was from Aldrich. pkMCADH was purified essentially as described by Lau et al (1986); however, Q-Sepharose (instead of Whatman DEAE-cellulose) and commercial calcium phosphate gel cellulose (Fluka) were used. The A278/446 ratio of the purified enzyme was = 5 and its activity comparable to that reported by Thorpe et al (1979).…”

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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“…The enzyme also exhibits an intrinsic (de)hydratase activity which is due to the capacity of the enzyme to abstract a proton reversibly from the a position, thus facilitating expulsion of HO-from the position [48].…”

Section: Substvates Undergoing A$-dehydrogenationmentioning

confidence: 99%

Mechanisms of flavoprotein-catalyzed reactions

Ghisla¹,

Massey²

1989

EJB Reviews 1989

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Flavoproteins are a class of enzymes catalyzing a very broad spectrum of redox processes by different chemical mechanisms. This review describes the best studied of these mechanisms and discusses factors possibly governing reactivity and specificity.A large number of flavin-containing enzymes, several hundreds, has been uncovered to date. An unusual feature of flavoproteins is the variety of the catalytic reactions performed, which range from typical redox catalysis such as the dehydrogenation of an amino acid, or the activation of dioxygen, to photochemistry; from 'DNA damage repair' to light emission. These few examples illustrate the fact that the same coenzyme is able to catalyze, or take part in catalytic events which must vary widely from a mechanistic point of view. The chemistry underlying the conversion itself will be quite different from case to case. This versatility sets flavoproteins apart from most other cofactor-dependent enzymes, which, in general, each catalyze a single type of chemical reaction. The activation or 'steering' of a particular activity of the flavin results from the interaction with the protein at the active center. On the other hand, for the vast majority of these enzymes a common feature exists, that at some stage during the catalytic event a transfer of electrons takes place between the substrate and the flavin itself.The purpose of the present review is not to enumerate the different functions of flavoproteins, this having been done elsewhere [l -41, but to focus on the mechanisms of catalysis, and on the possible ways of interaction of the flavin nucleus with the protein, i.e. on how this brings about chemistry appropriate to the particular enzyme reaction. In order toCorrespondence to S. Ghisla,

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

Cited by 45 publications

References 45 publications

Studies on the reaction mechanism of general acyl‐CoA dehydrogenase

Studies on the reaction mechanism of general acyl‐CoA dehydrogenase

Medium-Chain Acyl CoA Dehydrogenase: Evidence for Phosphorylation

Mechanisms of flavoprotein-catalyzed reactions

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