ON THE ROLE OF GLU376 IN CATALYSIS OF ACYL-CoA DEHYDROGENASES

Ankele, Kathrin; Melde, Klaus; Engst, Stefan; Bross, Peter; Ghisla, Sandro; Strauss, Arnold W.

doi:10.1515/9783110855425-063

Cited by 2 publications

(3 citation statements)

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“…5 %). Activity could not be recovered upon ultrafiltration, in contrast to what was reported by Ankele et al (1991) for the beef liver enzyme and approximately 40% of the theoretically possible amount of free CoA-SH was found in the ultrafiltrate by HPLC analysis. In analogy to the results by Thorpe's group with pkMCADH (Freund et al, 1985), this is compatible with turnover of the inactivator and with covalent modification of the protein.…”

Section: Resultscontrasting

confidence: 94%

“…The results obtained with 2-octynoyl-CoA and hwtLCADH or pkLCADH agree with the irreversible inactivation reported for MCADH (Freund et al, 1985). They contrast with those reported earlier by our group for beef liver LCADH (Ankele et al, 1991) which were interpreted as reflecting the absence of covalent protein modification. It should be noted that hwtLCADH (this work), hwtMCADH (Nandy et al, 1996b;Peterson et al, 1995) and a chimeric human MCADH which has the active-site Glu at position 261 instead of 376 (MCADH numbering; Nandy et al, 1996b) all differ substantially in their mode of reaction with 2-octynoyl-CoA.…”

Section: Discussionsupporting

confidence: 86%

“…Addition of this analogue to hwtLCADH at pH 7.6 (Kd approximately 170 pM) induces primarily a perturbation of the oxidized flavin spectrum (data not shown) consistent 2-Octynoyl-CoA inactivates medium-chain-acyl-CoA dehydrogenase rapidly, stoichiometrically and irreversibly, secondary to covalent reaction with the active-site base, E376-COO- (Freund et al, 1985;Powell and Thorpe, 1988). In contrast to this, it was reported that 2-octynoyl-CoA inactivates beef liver long-chain-acyl-CoA dehydrogenase reversibly without covalent modification of the protein moiety (Ankele et al, 1991). In the cases of pkLCADH and hwtLCADH, addition of 1 inol 2-octynoyl-CoA/mol to the oxidized enzymes leads to a rapid (kobs = 1.2 min-') formation of a long-wavelength band, centered at 562 nm (Fig.…”

Section: Resultsmentioning

confidence: 90%

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Characterization of Human and Pig Kidney Long‐Chain‐Acyl‐CoA Dehydrogenases and their Role in β‐Oxidation

Eder

Kräutle

Yü

et al. 1997

European Journal of Biochemistry

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Long-chain-acyl-CoA dehydrogenase (LCADH) has been produced by recombinant techniques from the human cDNA and purified after expression in Escherichia coli. Pig kidney LCADH was purified using an optimized method which also produces apparently pure short-chain-acyl-CoA dehydrogenase (SCADH) and medium-chain-acyl-CoA dehydrogenase (MCADH) in good yields. LCADH from both sources has a maximal turnover rate (V,,,,, of 650-700 min-' at pH 7.6) with the best substrates, which is approximately fivefold higher than reported previously. The human enzyme has an approximately fivefold higher K,,, compared with the pig kidney enzyme with substrates of chain length from C,, to C,, and a significantly different dependence of V,,,,, on the chain length. Pig kidney LCADH has a similar V,,,,IK,, with C,, to C,, substrates as MCADH does with C, to C,, substrates. Recombinant human LCADH, however, is significantly less efficient (approximately fourfold with C,J than purified pig kidney enzyme. We conclude that human LCADH is either quantitatively less important in P-oxidation than in the pig, or that post-translational modifications, not present in the recombinant human enzyme, are required to optimize human LCADH activity. Our results demonstrate that LCADH is as important as the other acylCoA dehydrogenases in fatty acid oxidation at physiological, mitochondria1 pH with optimal substrates of chain length ClO-Cl4. The extent of the LCADH-flavin cofactor reduction observed with most substrates and the rate of the subsequent reoxidation with oxygen are markedly different from those found with human medium chain acyl-CoA dehydrogenase. Both LCADH are inactivated by the substrate analogue 2-octynoyl-CoA, possibly via covalent modification of GIu261, the active-site residue involved in deprotonation of the substrate (a)C-H.Keywords: &oxidation; long chain ; acyl-CoA : dehydrogenase ; electron-transferring flavoprotein.Long-chain-acyl-CoA dehydrogenase (LCADH) is a member of a family of enzymes (Beinert, 1963;Nandy et al., 1996a;Tanaka and Indo, 1992), which catalyze in sequence the desaturation of fatty acyl-CoA conjugates. It differs from the other dehydrogenases of the family in its specificity ranging from medium(C,)-to long-chain(C,,) acyl-CoA substrates as well as in important parts of its sequence (Nandy et al., 1996a). As in isovaleryl-CoA dehydrogenase, the specific glutamate (conjugate) base which functions in the abstraction of the substrate (a)C-H as a proton, i.e. in the initiation of catalysis, was determined to reside at position 261 (position 255 ; medium-chain-acyl-CoA dehydrogenase MCADH numbering ; Djordjevic et al., 1994 ; Mohsen and Vockley, 1995). This position is located on helix G (Kim et al., 1993) and is different from that encountered within Correspondence to S . Ghisla, Faculty of Biology, University of Kon- E-mail: sandro.ghisla@uni-konstanz.deAbbreviations. C,-CoA, straight chain acyl-CoA thioesters, where X denotes the length of the carbon chain; hwt-and pkMCADH, human wild-type and pig kidney medium-...

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Section: Resultscontrasting

confidence: 94%

Section: Discussionsupporting

confidence: 86%

Section: Resultsmentioning

confidence: 90%

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Characterization of Human and Pig Kidney Long‐Chain‐Acyl‐CoA Dehydrogenases and their Role in β‐Oxidation

Eder

Kräutle

Yü

et al. 1997

European Journal of Biochemistry

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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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ON THE ROLE OF GLU376 IN CATALYSIS OF ACYL-CoA DEHYDROGENASES

Cited by 2 publications

References 1 publication

Characterization of Human and Pig Kidney Long‐Chain‐Acyl‐CoA Dehydrogenases and their Role in β‐Oxidation

Characterization of Human and Pig Kidney Long‐Chain‐Acyl‐CoA Dehydrogenases and their Role in β‐Oxidation

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

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