Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.3.99.3 (acyl-CoA dehydrogenase)
 1,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thia- and oxaoctanoyl-CoA derivatives (substituted at the C-3 and C-4 positions) have been synthesized to prove the reductive half-reaction in the medium-chain acyl-CoA dehydrogenase from pig kidney. 3-Thiaoctanoyl-CoA binds to this flavoenzyme, forming an intense, stable, long-wavelength band (at 804 nm; extinction coefficient = 8.7 mM-1 cm-1 at pH 7.6). The intensity of this band increases about 20% from pH 6.0 to pH 8.8. This long-wavelength species probably represents a charge-transfer complex between bound acyl enolate as the donor and oxidized flavin adenine dinucleotide as the acceptor. Thus, the enzyme catalyzes alpha-proton exchange, and no long-wavelength bands are seen with 3-thiaoctyl-CoA (where the carbonyl moiety is replaced by a methylene group). 3-Oxaoctanoyl-CoA binds comparatively weakly to the dehydrogenase, with a long-wavelength band at 780 nm which is both less intense and less stable than the corresponding thia analogue. These data suggest that the enzyme can accomplish alpha-proton abstraction from certain weakly acidic acyl-CoA derivatives, without concerted transfer of a hydride equivalent to the flavin. 4-Thiaoctanoyl-CoA is dehydrogenated in the standard assay 1.5-fold faster than octanoyl-CoA. Titrations of the medium-chain dehydrogenase with the 4-thia derivative resemble those obtained with octanoyl-CoA, except for the contribution of the strongly absorbing 4-thia-trans-2-octenoyl-CoA product. The corresponding 4-oxa analogue is a much poorer substrate (10% of the rate shown by octanoyl-CoA) but again effects substantially complete reduction of the flavin chromophore in the dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The reductive half-reaction in Acyl-CoA dehydrogenase from pig kidney: studies with thiaoctanoyl-CoA and oxaoctanoyl-CoA analogues. 316 33

The mechanisms of the initial interactions of three rat liver acyl-CoA dehydrogenases (short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases) and their fatty acyl-CoA substrate were studied using enzyme-catalyzed deuterium exchange. The reaction products were identified and quantitated using mass spectroscopy and 1H-NMR. When fatty acyl-CoA substrates were incubated with catalytic amounts of acyl-CoA dehydrogenase in D2O in the absence of an electron acceptor, a rapid monodeuteration of the substrate occurred to replace one of the prochiral C-2 hydrogens, while no C-3 hydrogens were exchanged with deuterium. The C-2 monodeuteration proceeded to the extent of 80% of the total amount of substrate added at 90 min and almost to completion at 120 min. The pKa values and optimum pD values for the C-2 proton/deuteron exchange reactions were 6.0 and 7.5, respectively, for each of the three acyl-CoA dehydrogenases. The apparent turnover numbers were 3.0, 3.3, and 0.5 s-1 for short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases, respectively. These results provide the first direct evidence for carbanion formation via abstraction of a C-2 hydrogen by a base in the enzyme, as the first step of the catalytic pathway of acyl-CoA dehydrogenation. When the acyl-CoA dehydrogenases were reacted with moderate excesses of acyl-CoA substrates in D2O in the absence of an electron acceptor, maximum bleaching of the FAD absorbance and the appearance of the long wavelength absorbance, attributed to a charge transfer complex, were observed. However, the dehydrogenation products, 2-enoyl-CoAs, were produced either not at all or in an amount which represented only a minor fraction of the amount of the enzyme added, while the substrates in the enzyme-substrate complexes rapidly turned over as indicated by the extensive monodeuteration which concomitantly occurred. Unlike previous hypothesis, these results indicate that the hydride ion transfer from C-3 of the substrate to the enzyme-FAD is not yet complete in the charge-transfer complex. The transfer of the hydride ion to alloxazine N-5 and the release of products are completed only in the presence of electron-transfer flavoprotein or another suitable electron acceptor.
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PMID:Mechanism of action of short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases. Direct evidence for carbanion formation as an intermediate step using enzyme-catalyzed C-2 proton/deuteron exchange in the absence of C-3 exchange. 396 64

Pig kidney general acyl-CoA dehydrogenase is rapidly, stoichiometrically, and irreversibly inactivated by the acetylenic thio ester 2-octynoyl coenzyme A (2-octynoyl-CoA). The inhibitor binds initially to the dehydrogenase with a 10-nm red shift and increased resolution of the flavin chromophore, followed by the generation of a charge-transfer complex between some form of the bound inhibitor and oxidized flavin (lambda max 800 nm; epsilon app = 4.5 mM-1 cm-1; k1 = 1.07 min-1, at pH 7.6, 25 degrees C). The rate of formation of the long wavelength band is increased markedly with increasing pH (pKapp = 7.9). This intermediate then decays with release of about 0.6 mol of CoASH at pH 7.6, yielding a final form with a spectrum typical of bound oxidized flavin. Both irreversible inactivation and covalent modification of the protein occur prior to the decay of the long wavelength species. The modified dehydrogenase is not reduced on prolonged anaerobic incubation with the substrate octanoyl-CoA. The inactive enzyme is unusually resistant to dithionite reduction but may be readily photoreduced via the blue semiquinone to the dihydroflavin form. This reduced enzyme is rapidly reoxidized by electron-transferring flavoprotein, the physiological electron acceptor of the dehydrogenase. General acyl-CoA dehydrogenase is also inactivated by 2-pentynoyl- and 2-pentadecynoyl-CoA with formation of an 800-nm band of lower intensity and by propiolyl-CoA, phenylpropiolyl-CoA, and 2-octynoylpantetheine without the appearance of detectable intermediate species. These data are compared with the behavior of acyl-CoA dehydrogenases toward mechanism-based inactivators carrying an acetylene function at C-3, e.g., 3-butynoyl-CoA.
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PMID:Inactivation of general acyl-CoA dehydrogenase from pig kidney by 2-alkynoyl coenzyme A derivatives: initial aspects. 408 3

Aspects of the binding and dehydrogenation of acyl-CoA thiol esters by the general acyl-CoA dehydrogenase from pig liver were investigated using a dead-end inhibitor, S-octyl-CoA, several alternate substrates, and three active site-directed inhibitors. Experiments with S-octyl-CoA indicate that the carbonyl group of acyl-CoA thiol esters is not absolutely required for binding to the enzyme. However, the mode of binding of the 8-carbon thiol ether can be distinguished from the mode of binding of the enoyl-CoA product, octenoyl-CoA. Octanoyl pantetheine, octanoyl-etheno-CoA, and octanoyl-3'-dephospho-CoA are alternate substrates of the dehydrogenase. Steady state kinetic constants obtained with these alternate substrates indicate that the adenosine 5'-diphosphate, but not the 3'-phosphate, of the nucleotide moiety of acyl-CoA substrates contribute to the tight binding of the substrates. The substrate analogs 3'-butynoyl-CoA and 3-octynoyl-CoA are active site-directed, mechanism-based irreversible inhibitors of the dehydrogenase. These inhibitors covalently modify the apoprotein rather than the flavin. This finding and the fact that 2,3-octadienoyl-CoA also completely and irreversibly inhibits the enzyme indicate that th 3-acetylenic thiol esters inhibit the enzyme by a mechanism involving: (1) base-catalyzed abstraction of a protein at C-2 followed by isomerization to the allene carbanion, (2) protonation of the carbanion, and (3) attack of a nucleophile in the enzyme-active site on C-3 of the 2,3-dienoyl-CoA. The data show that the alkynoyl-CoA's are activated and bound at the active site of the enzyme. The results suggest that abstraction of a proton at C-2 of acyl-CoA substrates is the initial step in the catalytic pathway of dehydrogenation of substrates by the enzyme.
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PMID:Enzyme-activated inhibitors, alternate substrates, and a dead end inhibitor of the general acyl-CoA dehydrogenase. 744 May 36

Medium chain acyl-CoA dehydrogenase from pig kidney catalyzes the oxidation of acyl-CoA thioesters to trans-2-enoyl-CoA derivatives with an optimal chain length of about C-8. The binding energy for alkyl-SCoA thioethers shows no such optimum but increases linearly from C-2 to C-16 with a slope of about 390 cal/-CH2 group. In contrast, four types of CoA-thioester analogues (2-aza-acyl-, 3-thia-acyl-, 3-keto-acyl-, and trans-2-enoyl-) yield an incremental binding energy of about 800 cal/-CH2 group until a chain length of about C-8 is reached. The observed binding energy then decreases, or remains constant, with increasing chain length. Studies with dithiooctanoyl-CoA and 2-azadithiooctanoyl-CoA show that the C = S moiety is accommodated poorly by the medium chain dehydrogenase. A model for chain length discrimination, based on the crystal structure of the enzyme [Kim, J. J. P., Wang, M., & Paschke, R. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7523-7527], is proposed in which hydrogen-bonding interactions between enzyme and thioester carbonyl oxygen atom are maximized at optimal chain lengths. Oversized chains decrease the frequency of effective alignment between enzyme and the C-1 to C-3 region of thioester ligands. Thus the extent of polarization of bound 4-thia-trans-2-enoyl-CoA thioesters decreases sharply with chains longer than C-12.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Role of the carbonyl group in thioester chain length recognition by the medium chain acyl-CoA dehydrogenase. 761 1

D-kijanose is an unusual nitrosugar found attached to the antibiotic kijanimicin. Ten enzymes are required for its production in Actinomadura kijaniata, a soil-dwelling actinomycete. The focus of this investigation is on the protein encoded by the kijd3 gene and hereafter referred to as KijD3. On the basis of amino acid sequence analyses, KijD3 has been proposed to be an FAD-dependent oxidoreductase, which catalyzes the sixth step in d-kijanose biosynthesis by converting dTDP-3-amino-2,3,6-trideoxy-4-keto-3-methyl-d-glucose into its C-3' nitro derivative. This putative activity, however, has never been demonstrated in vivo or in vitro. Here we report the first structural study of this enzyme. For our investigation, crystals of KijD3 were grown in the presence of dTDP, and the structure was solved to 2.05-A resolution. The enzyme is a tetramer with each subunit folding into three distinct regions: a five alpha-helical bundle, an eight-stranded beta-sheet, and a second five alpha-helical bundle. The dTDP moiety is anchored to the protein via the side chains of Glu 113, Gln 254, and Arg 330. The overall fold of KijD3 places it into the well-characterized fatty acyl-CoA dehydrogenase superfamily. There is a decided cleft in each subunit with the appropriate dimensions to accommodate a dTDP-linked sugar. Strikingly, the loop defined by Phe 383 to Ala 388, which projects into the active site, contains two adjacent cis-peptide bonds, Pro 386 and Tyr 387. Activity assays demonstrate that KijD3 requires FAD for activity and that it produces a hydroxylamino product. The molecular architecture of KijD3 described in this report serves as a paradigm for a new family of enzymes that function on dTDP-linked sugar substrates.
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PMID:X-ray structure of kijd3, a key enzyme involved in the biosynthesis of D-kijanose. 2033 31