On the Mechanism of Dehydrogenation of Fatty Acyl Derivatives of Coenzyme A

Crane, F.L.; Mii, Shinsuke; Hauge, Jens G.; Green, D. E.; Beinert, Helmut

doi:10.1016/s0021-9258(18)65836-3

Cited by 140 publications

(28 citation statements)

References 22 publications

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“…[R-2 H 2 ]-4NPA-CoA was used for a kinetic verification, via measurement of a deuterium isotope effect, of the assumption that the observed spectral changes are directly connected with the abstraction of the ligand RC-H. Incubation of 4NPA-CoA in D 2 O buffered with 10 mM NaP i , pD 7.1, leads to >90% exchange of the RC-H with deuterium within 20 min, as can be verified by 1 H NMR spectroscopy (data not shown). While this rate is insignificant compared to the enzymecatalyzed rate of deprotonation, it does not allow experimentation with deuterated substrate in H 2 O due to the time required for sample preparation.…”

Section: Kinetics Of Enzyme Interaction With the Ligands: (A) [R-2 H ...mentioning

confidence: 92%

“…The aryl bromide, 10 mmol dissolved in 40 mL of dry DMF under N 2 , was heated at 100 °C with 10 mmol of Cu(I) iodide and 50 mmol of sodium acetylacetonate to afford the corresponding (3arylacetyl)acetonate [3 h for (4-cyanophenyl)acetone and 15 h for (4-acetylphenyl)acetone]. The formed product was subsequently deacetylated by addition of 40 mL of 1 M NaOH at ambient temperature and incubation for 3 h. After filtration and extraction with methylene chloride, the crude product was purified by silica gel flash chromatography (CH 2 Cl 2 /ethyl acetate 9:1) and identified by 1 H NMR and fast atom bombardment mass spectroscopy [(4-cyanophenyl)acetone mp 51 °C, yield 35%; (4-acetylphenyl)acetone mp 34 °C, yield 45%).…”

Section: Methodsmentioning

confidence: 99%

“…A peculiarity of the R,β-dehydrogenation reaction is that it involves the concomitant fission of two kinetically stable C-H bonds. In the past, studies with medium-chain acyl-CoA dehydrogenase (MCADH) 1 have led to considerable insight concerning the mechanism of this reaction. It proceeds via an anti elimination where the pro(R)-R-proton is abstracted by the active-site base Glu376, and the β-hydrogen (3,4) is transferred as hydride to the flavin N(5) position (5).…”

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confidence: 99%

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Substrate Activation by Acyl-CoA Dehydrogenases: Transition-State Stabilization and pKs of Involved Functional Groups

et al. 1998

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ABSTRACT:The mechanism by which acyl-CoA dehydrogenases initiate catalysis was studied by using p-substituted phenylacetyl-CoAs (substituents -NO 2 , -CN, and CH 3 CO-), 3S-C 8 -, and 3′-dephospho-3S-C 8 CoA. These analogues lack a C-H and cannot undergo R, -dehydrogenation. Instead they deprotonate at RC-H at pH g 14 to form delocalized carbanions having strong absorbancies in the near UV-visible spectrum. The pK a s of the corresponding phenylacetone analogues were determined as ≈13.6 (-NO 2 ), ≈14.5 (-CN), and ≈14.6 (CH 3 CO-). Upon binding to human wild-type medium-chain acyl-CoA dehydrogenase (MCADH), all analogues undergo RC-H deprotonation. While the extent of deprotonation varies, the anionic products form charge-transfer complexes with the oxidized flavin. From the pH dependence of the dissociation constants (K d ) of p-NO 2 -phenylacetyl-CoA (4NPA-CoA), 3S-C 8 -CoA, and 3′-dephospho-3S-C 8 CoA, four pK a s at ≈5, ≈6, ≈7.3, and ≈8 were identified. They were assigned to the following ionizations: (a) pK a ≈5, ligand (L-H) in the MCADH∼ligand complex; (b) pK a ≈6, Glu376-COOH in uncomplexed MCADH; (c) pK a ≈7.3, Glu99-COOH in uncomplexed MCADH (Glu99 is a residue that flanks the bottom of the active-center cavity; this pK is absent in the mutant Glu99Gly-MCADH); and (d) pK ≈8, Glu99-COOH in the MCADH∼4NPA-CoA complex. The pK a ≈6 (b) is not significantly affected in the MCADH∼4NPA-CoA complex, but it is increased by g1 pK unit in that with 3S-C 8 CoA and further in the presence of C 8 -CoA, the best substrate. The RC-H pK a s of 4NPA-CoA, of 3S-C 8 -CoA, and of 3′-dephospho-3S-C 8 CoA in the complex with MCADH are ≈5, ≈5, and ≈6. Compared to those of the free species these pK a values are therefore lowered by 8 to g11 pH units (50 to g 65 kJ mol -1 ) and are close to the pK a of Glu376-COOH in the complex with substrate/ligand. This effect is ascribed mainly to the hydrogen-bond interactions of the thioester carbonyl group with the ribityl-2′-OH of FAD and Glu376-NH. It is concluded that the pK a shifts induced with normal substrates such as n-octanoyl-CoA are still higher and of the order of 9-13 pK units. With 4NPA-CoA and MCADH, RC-H abstraction is fast (k app ≈55 s -1 at pH 7.5 and 25°C, deuterium isotope effect ≈1.34). However, it does not proceed to completion since it constitutes an approach to equilibrium with a finite rate for reprotonation in the pH range 6-9.5. The extent of deprotonation and the respective rates are pH-dependent and reflect apparent pK a s of ≈5 and ≈7.3, which correspond to those determined in static experiments.Acyl-CoA dehydrogenases catalyze the R, -dehydrogenation of fatty acid acyl-CoA conjugates to their corresponding enoyl-CoA products; the redox equivalents formed in this reaction are transferred to electron transferring flavoprotein and further to the respiratory chain (1, 2). A peculiarity of the R, -dehydrogenation reaction is that it involves the concomitant fission of two kinetically stable C-H bonds. In the past, studies with medium-chain acyl-CoA dehydrogenase ...

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Section: Kinetics Of Enzyme Interaction With the Ligands: (A) [R-2 H ...mentioning

confidence: 92%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Substrate Activation by Acyl-CoA Dehydrogenases: Transition-State Stabilization and pKs of Involved Functional Groups

et al. 1998

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“…Enzyme Assay. Acyl-CoA dehydrogenase activity was followed spectrophotometrically, by using 2,6-dichlorophenolindophenol (DCPIP) as the terminal electron acceptor (Crane et al, 1956;Ikeda et al, 1985), by the ferricenium assay (Lehman & Thorpe, 1990), or by following the decrease in fluorescence of electron-transfer flavoprotein (ETF) (Beckman & Frerman, 1983). The reaction was initiated by addition of the purified enzyme or, when crude cell extract was being assayed, by addition of the fatty acyl-CoA substrates (Sigma).…”

Section: Methodsmentioning

confidence: 99%

Identification of the Catalytic Base in Long Chain Acyl-CoA Dehydrogenase

Djordjević¹,

Yü²,

Paschke³

et al. 1994

Biochemistry

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We have used molecular modeling and site-directed mutagenesis to identify the catalytic residues of human long chain acyl-CoA dehydrogenase. Among the acyl-CoA dehydrogenases, a family of flavoenzymes involved in beta-oxidation of fatty acids, only the three-dimensional structure of the medium chain fatty acid specific enzyme from pig liver has been determined (Kim, J.-J.P., Wang, M., & Paschke, R. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7523-7527). Despite the overall sequence homology, the catalytic residue (E376) of medium chain acyl-CoA dehydrogenase is not conserved in isovaleryl- and long chain acyl-CoA dehydrogenases. A molecular model of human long chain acyl-CoA dehydrogenase was derived using atomic coordinates determined by X-ray diffraction studies of the pig medium chain specific enzyme, interactive graphics, and molecular mechanics calculations. The model suggests that E261 functions as the catalytic base in the long-chain dehydrogenase. An altered dehydrogenase in which E261 was replaced by a glutamine was constructed, expressed, purified, and characterized. The mutant enzyme exhibited less than 0.02% of the wild-type activity. These data strongly suggest that E261 is the base that abstracts the alpha-proton of the acyl-CoA substrate in the catalytic pathway of this dehydrogenase.

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“…IBD is responsible for the third step in the breakdown of valine at which it converts isobutyryl-CoA into methylacrylyl-CoA [1,2] . ACADs are a family of nuclear encoded, mitochondrial flavoenzymes that catalyze the , -desaturation of acyl-CoA to corresponding trans-2-enoyl-CoA in the catabolism of fatty acids and branched chain amino acids [3][4][5][6][7] . ACADs are classified according to the acyl moieties in their substrates into two subclasses.…”

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confidence: 99%

Stopped-flow kinetics and cysteine residue protonation of human isobutyryl-CoA dehydrogenase

2017

Egyptian Journal of Pure and Applied Science

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Human Isobutyryl-CoA Dehydrogenase, IBD, is involved in the valine amino acid catabolism. It catalyzes with a high specificity the reduction of the isobutyryl-CoA to methylacrylyl-CoA in contrary to the rat Short-Branched Chain Acyl-CoA Dehydrogenase, SBCAD, which is able to utilize both 2methylbutyryl-CoA and isobutyryl-CoA that emerged from Ile and Val degradation, respectively. Here, the IBD stopped-flow kinetic was studied and it was found that IBD reduction reaction has two phases, the faster one was exponential at pH 9.0 while it was much slower and multi exponential in pH 6.5. Also, it was found that isobutyryl-CoA binds IBD in two steps, where the first step was the faster one. The protonation of the high IBD Cys content was computed by the H++ server and the results are discussed. In conclusion, one or more of Cys residues interact and involved in the pKa value of the active site Glu-376. It could thus be concluded that Cys residue(s) has crucial role(s) in the conformation stability and biocatalytic activity of the IBD.

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On the Mechanism of Dehydrogenation of Fatty Acyl Derivatives of Coenzyme A

Cited by 140 publications

References 22 publications

Substrate Activation by Acyl-CoA Dehydrogenases: Transition-State Stabilization and pKs of Involved Functional Groups

Substrate Activation by Acyl-CoA Dehydrogenases: Transition-State Stabilization and pKs of Involved Functional Groups

Identification of the Catalytic Base in Long Chain Acyl-CoA Dehydrogenase

Stopped-flow kinetics and cysteine residue protonation of human isobutyryl-CoA dehydrogenase

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