Structure-function correlation of fatty acyl-CoA dehydrogenase and fatty acyl-CoA oxidase (original) (raw)

Oxidative Inactivation of a Charge Transfer Complex in the Medium-Chain Acyl-CoA Dehydrogenase

Biochemistry, 1995

The intense charge transfer complex between the enolate of 3-thia-octanoyl-CoA and the oxidized flavin of the medium-chain acyl-CoA dehydrogenase is discharged by the femcenium ion with irreversible inactivation of the enzyme. Charge transfer complex formation is a necessary, but insufficient, condition for oxidative inactivation: the 3-oxa-octanoyl-CoA complex is also inactivated, whereas the comparable trans-3-octenoyl-CoA species is not. Complete inactivation of the dehydrogenase with 3-thiaoctanoyl-CoA requires 1 molecule of thioester and apparently 3 molecules of ferricenium hexafluorophosphate. Experiments with 8-Cl-FAD substituted enzyme and the crystal structure of enzyme-ligand complexes argue that ferricenium ion-mediated oxidation proceeds through the flavin prosthetic group.

Kinetic methods for the study of the enzyme systems of β-oxidation

Archives of Biochemistry and Biophysics, 1983

Kinetic methods for studying the reactions of the "general" fatty acyl CoA dehydrogenase under three sets of substrate and enzyme concentration conditions have been developed. The reaction of butyryl-CoA and electron transfer flavoprotein (ETF) can be studied either under steady-state conditions with enzyme at catalytic concentration or under single-turnover conditions with enzyme in excess. Under the latter conditions, acyl-CoA dehydrogenase acts both as a catalyst and an ultimate electron-transfer acceptor. The reductive half-reaction of butyryl-CoA and enzyme can also be studied in a separate kinetic experiment. Comparison of the pH dependences of the rate constants and isotope effects of the steady-state reaction of butyryl-CoA and ETF with the same parameters for the reductive half-reaction is consistent with a mechanism involving transfer of electrons from butyryl-CoA to ETF within a ternary complex. An alternative mechanism in which the reductive half-reaction takes place prior to the binding and reaction of ETF seems unlikely because the pH 8.5 isotope effect on the reductive halfreaction is much larger than that on the complete reaction in spite of the fact that the rates of the reactions are comparable. The pH dependence of the Km for substrate and KI for inhibitor is consistent with a mechanism for transfer of electrons within the ternary complex which involves protonation of the C=O group of substrates. The protonation labilizes the C-2 proton and base catalysis of the removal of the C-2 proton results in the production of the active enzyme-substrate species, namely the C-2 anion of substrate. Early investigations on fatty acyl-CoA dehydrogenase led to the discovery that there are three enzymes involved in the first step of the P-oxidation pathway (l-3). Each of these has different specificity, resulting in the characterization of a short-chain, a "general," and a long-chain fatty acyl-CoA dehydrogenase. With the

Oxidase Activity of the Acyl-CoA Dehydrogenases †

Biochemistry, 1998

The medium chain acyl-CoA dehydrogenase catalyzes the flavin-dependent oxidation of a variety of acyl-CoA thioesters with the transfer of reducing equivalents to electron-transferring flavoprotein. The binding of normal substrates profoundly suppresses the reactivity of the reduced enzyme toward molecular oxygen, whereas the oxidase reaction becomes significant using thioesters such as indolepropionyl-CoA (IP-CoA) and 4-(dimethylamino)-3-phenylpropionyl-CoA (DP-CoA). Steady-state and stopped-flow studies with IP-CoA led to a kinetic model of the oxidase reaction in which only the free reduced enzyme reacts with oxygen (Johnson, J. K., Kumar, N. R., and Srivastava, D. K. (1994) Biochemistry 33, 4738-4744). We have tested their proposal with IP-CoA and DP-CoA. The dependence of the oxidase reaction on oxygen concentration is biphasic with a major low affinity phase incompatible with a model predicting a simple K m for oxygen of 3 µM. If only free reduced enzyme reacts with oxygen, increasing IP-CoA would show strong substrate inhibition because it binds tightly to the reduced enzyme. Experimentally, IP-CoA shows simple saturation kinetics. The Glu376-Gln mutant of the medium chain dehydrogenase allows the oxygen reactivity of complexes of the reduced enzyme with IP-CoA and the corresponding product indoleacryloyl-CoA (IA-CoA) to be characterized without the subsequent redox equilibration that complicates analysis of the oxidase kinetics of the native enzyme. In sum, these data suggest that when bulky, nonphysiological substrates are employed, multiple reduced enzyme species react with molecular oxygen. The relatively high oxidase activity of the short chain acyl-CoA dehydrogenase from the obligate anaerobe Megasphaera elsdenii was studied by rapid reaction kinetics of wild-type and the Glu367-Gln mutant using butyryl-, crotonyl-, and 2-aza-butyryl-CoA thioesters. In marked contrast to those of the mammalian dehydrogenase, complexes of the reduced bacterial enzyme with these ligands react with molecular oxygen at rates similar to those of the free protein. Evolutionary and mechanistic aspects of the suppression of oxygen reactivity in the acyl-CoA dehydrogenases are discussed.

Inactivation of two-electron reduced medium chain acyl-CoA dehydrogenase by 2-octynoyl-CoA*1

Archives of Biochemistry and Biophysics, 1989

The acetylenic thioester, 2-octynoyl-CoA, inactivates medium chain acyl-CoA dehydrogenase from pig kidney by two distinct pathways depending on the redox state of the FAD prosthetic group. Inactivation of the oxidized dehydrogenase occurs with labeling of an active site glutamate residue and elimination of CoASH. Incubation of the reduced dehydrogenase with 2-octynoyl-CoA rapidly forms a kinetically stable dihydroflavin species which is resistant to reoxidation using trans-2-octenoyl-CoA, molecular oxygen, or electron transferring flavoprotein. The reduced enzyme derivative shows extensive bleaching at 446 nm with shoulders at 320 and 380 nm. Denaturation of the reduced derivative in 80% methanol yields a mixture of products which was characterized by HPLC, by uv/vis, and by radiolabeling experiments. Approximately 20% of the flavin is recovered as oxidized FAD, about 40% is retained covalently attached to the protein, and the remainder is distributed between several species eluting after FAD on reversephase HPLC. The spectrum of one of these species ressembles that of a N(5)-C(4a) dihydroflavin adduct. These data suggest that a primary reduced flavin species undergoes various rearrangements during release from the protein. The possibility that the inactive modified enzyme represents a covalent adduct between 2-octynoyl-CoA and reduced flavin is discussed. Analogous experiments using enzyme substituted with 1,5-dihydro-5deaza-FAD show rapid and quantitative reoxidation of the flavin by 0.5 eq of 2-octynoyl-CoA. L' 19X9 Ararlcmic Press, Inc. Acyl-CoA dehydrogenases are flavoproteins catalyzing the oxidation of acyl-CoA thioesters to their corresponding tram-2enoyl-CoA derivatives (1):

Inactivation of two-electron reduced medium chain acyl-CoA dehydrogenase by 2-octynoyl-CoA

Archives of Biochemistry and Biophysics, 1989

Kinetic and spectral properties of isovaleryl-CoA dehydrogenase and interaction with ligands

Biochimie, 2015

Isovaleryl-CoA dehydrogenase (IVD) catalyzes the conversion of isovaleryl-CoA to 3methylcrotonyl-CoA and the transfer of electrons to the electron transfer flavoprotein (ETF). Recombinant human IVD purifies with bound CoA-persulfide. A modified purification protocol was developed to isolate IVD without bound CoA-persulfide and to protect the protein thiols from oxidation. The CoA-persulfide-free IVD specific activity was 112.5 µmol porcine ETF•min −1 •mg −1 , which was ~20-fold higher than that of its CoA-persulfide bound form. The K m and catalytic efficiency (k cat /K m) for isovaleryl-CoA were 1.0 µM and 4.3 × 10 6 •M −1 •sec −1 per monomer, respectively, and its K m for ETF was 2.0 µM. Anaerobic titration of isovaleryl-CoA into an IVD solution resulted in a stable blue complex with increased absorbance at 310 nm, decreased absorbance at 373 and 447 nm, and the appearance of the charge transfer complex band at 584 nm. The apparent dissociation constant (K D app) determined spectrally for isovaleryl-CoA was 0.54 µM. Isovaleryl-CoA, acetoacetyl-CoA, methylenecyclopropylacetyl-CoA, and ETF induced CD spectral changes at the 250-500 nm region while isobutyryl-CoA did not, suggesting conformational changes occur at the flavin ring that are ligand specific. Replacement of the IVD Trp166 with a Phe did not block IVD interaction with ETF, indicating that its indole ring is not essential for electron transfer to ETF. A twelve amino acid synthetic peptide that matches the sequence of the ETF docking peptide competitively inhibited the enzyme reaction when ETF was used as the electron acceptor with a K i of 1.5 mM. Keywords acyl-CoA dehydrogenase; electron-transferring flavoprotein Isovaleryl-CoA dehydrogenase (IVD; 1 EC 1.3.99.10) is an intramitochondrial homotetrameric flavoenzyme in the leucine catabolism pathway that catalyzes the α,β

Catalysis of electron transfer during activation of O 2 by the flavoprotein glucose oxidase

Proceedings of the National Academy of Sciences, 2002

Two prototropic forms of glucose oxidase undergo aerobic oxidation reactions that convert FADH − to FAD and form H 2 O 2 as a product. Limiting rate constants of k cat / K M (O 2 ) = (5.7 ± 1.8) × 10 2 M −1 ⋅s −1 and k cat / K M (O 2 ) = (1.5 ± 0.3) × 10 6 M −1 ⋅s −1 are observed at high and low pH, respectively. Reactions exhibit oxygen-18 kinetic isotope effects but no solvent kinetic isotope effects, consistent with mechanisms of rate-limiting electron transfer from flavin to O 2 . Site-directed mutagenesis studies reveal that the pH dependence of the rates is caused by protonation of a highly conserved histidine in the active site. Temperature studies (283–323 K) indicate that protonation of His-516 results in a reduction of the activation energy barrier by 6.0 kcal⋅mol −1 (0.26 eV). Within the context of Marcus theory, catalysis of electron transfer is attributed to a 19-kcal⋅mol −1 (0.82 eV) decrease in the reorganization energy and a much smaller 2.2-kcal⋅mol −1 (0.095 eV) en...

Structure-function correlation of fatty acyl-CoA dehydrogenase and fatty acyl-CoA oxidase (original) (raw)

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