Inactivation of two-electron reduced medium chain acyl-CoA dehydrogenase by 2-octynoyl-CoA (original) (raw)
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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):
Biochemistry, 1988
Thia-and oxaoctanoyl-CoA derivatives (substituted at the C-3 and C-4 positions) have been synthesized to probe 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-' cm-' at p H 7.6). The intensity of this band increases about 20% from p H 6.0 to p H 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 a-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 a-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.
Studies with general acyl-CoA dehydrogenase from pig kidney
European Journal of Biochemistry, 2008
3,4-Pentadienoyl-CoA, an allenic substrate analog, is a potent inhibitor of the flavoprotein pig-kidney general acyl-CoA dehydrogenase. The analog reacts very rapidly (k = 2.4 x lo3 min-') with the native oxidized enzyme to form a covalent flavin adduct probably involving the isoalloxazine position N-5. This species is inactive, but activity may be regained by two pathways. The allenic thioester can be displaced ( k = 0.3 min-') by a large excess of octanoyl-CoA substrate upon reversal of covalent adduct formation. Alternatively, the enzyme inactivator adduct slowly decomposes (tljz = 75 min) to form the strongly thermodynamically favoured 2,4-diene and catalytically active, oxidized enzyme. During this latter process 15 -20% of the activity is irreversibly lost probably due to covalent modification of the protein. These data suggest that 3,4-pentadienoyl-CoA should be considered a suicide substrate of the acyl-CoA dehydrogenase. The mechanism of the reactions, and in particular the 3,4+2,4 tautomerization, are consistent with a catalytic sequence initiated by abstraction of an a-hydrogen as a proton.
Biochemistry, 1985
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 (A,,, 800 nm; eapp = 4.5 mM-' cm-'; kl = 1.07 mi&, at p H 7.6, 25 "C). The rate of formation of the long wavelength band is increased markedly with increasing p H (pKapP = 7.9). This intermediate then decays
Reaction of General Acyl-CoA Dehydrogenase with 3,4-Pentadienoyl-CoA
Recently several thioester inhibitors of acyl-CoA dehydrogenases have been described whose mode of action probably involves an initial removal of a C-2 proton followed by isomerization of the thioester to the active species. Thus general acyl-CoA dehydrogenase is inhibited via irreversible covalent modifica tion of the protein using 3-alkynoyl-CoA derivatives (1), and a similar inhibi tion has been reported for butyryl-CoA dehydrogenase from Megasphaera elsdenii and glutaryl-CoA dehydrogenase from Pseudomonas fluorescens using 3-alkynoyl-pantetheine thioesters (2). Methylenecyclopropylacetyl-CoA, a metabolite of hypoglycin, effects irreversible flavin modification on incubation with M. elsdenii butyryl-CoA dehydrogenase and pig kidney general acyl-CoA dehydrogenase (3). This paper deals with 3,4-pentadienoyl-CoA, a novel inhibitor of general acyl-CoA dehydrogenase. The mode of action of this allenic thioester is also consistent with an initial proton abstraction.
Archives of Biochemistry and Biophysics, 2001
The redox-inactive thioester analog 3-thia-octanoyl-CoA blocks transfer of a hydride equivalent to the flavin prosthetic group of the medium-chain acyl-CoA dehydrogenase with the accumulation of a stable enolate intermediate not encountered with normal substrates. Substitution of the normal flavin with 5-deaza-FAD would thus be expected to lead to enolate formation with both normal and 3-thiasubstrate analogs, because reduction of the 5-deazaenzyme is thermodynamically highly unfavorable. However, spectrophotometric titrations show that neither ligand forms significant enolate species with the 5-deaza-FAD enzyme. Similarly, the substituted dehydrogenase catalyzes undetectable ␣-proton exchange with octanoyl-CoA and ca. 1% of the corresponding rate with 3-thia-octanoyl-CoA when compared to the native enzyme. This inability to stabilize enolate species is not simply due to impaired binding of CoA-thioester analogs, because binding of a range of ligands is weakened by only 2-to 10-fold with the 5-deaza-enzyme. 4-Thia-trans-2-enoyl-CoA product is polarized normally on binding to the substituted protein, showing that this critical aspect of catalysis is apparently normal. These data, together with studies with CoA-persulfide and acetoacetyland p-nitrophenylacetyl-CoA, suggest that 5-deaza-FAD substitution exerts subtle, unanticipated, effects on the reductive half-reaction of the mediumchain acyl-CoA dehydrogenase. The involvement of charge-transfer interactions in the acidification of weakly acidic acyl-CoA thioesters is discussed.
Alternate electron acceptors for medium-chain acyl-CoA dehydrogenase: use of ferricenium salts
Biochemistry, 1990
Medium-chain acyl-CoA dehydrogenase reduced with octanoyl-CoA is reoxidized in two one-electron steps by two molecules of the physiological oxidant, electron transferring flavoprotein (ETF). The organometallic oxidant ferricenium hexafluorophosphate (Fc+PF6-) is an excellent alternative oxidant of the dehydrogenase and mimics a number of the features shown by ETF. Reoxidation of octanoyl-CoA-reduced enzyme (200 pM Fc'PFc in 100 m M Hepes buffer, pH 7.6, 1 "C) occurs in two one-electron steps with pseudo-first-order rate constants of 40 s-l and about 200 s-l for kl and k2, respectively. The reaction is comparatively insensitive to ionic strength, and evidence of rate saturation is encountered at high ferricenium ion concentration. As observed with ETF, the free two-electron-reduced dehydrogenase is a much poorer kinetic reductant of Fc+PF6-, with rate constants of 3 s-l and 0.3 s-l (for k l and k2, respectively) using 200 pM Fc' PF;.
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.