Inactivation of medium-chain acyl-CoA dehydrogenase by oct-4-en-2-ynoyl-CoA.

Mitochondrial medium-chain acyl-CoA dehydrogenase is a key enzyme for the beta-oxidation of fatty acids, which catalyzes the FAD-dependent oxidation of a variety of acyl-CoA substrates to the corresponding trans-2-enoyl-CoA thioesters. Oct-4-en-2-ynoyl-CoA was identified as a new irreversible inhibitor of acyl-CoA dehydrogenase, and kinetic parameters K(I) and k(inact) were determined to be 11 microM and 0.025 min(-1), respectively. Triple bond between C2 and C3 of the inhibitor was identified as the functional group responsible for enzyme inactivation, and Michael addition is proposed as the mechanism for this inactivation, which is a new pathway for inactivation of MCAD by inhibitors. The inhibitor may become a lead for further development for treating non-insulin-dependent diabetes mellitus.

Interaction of 3,4-dienoyl-CoA thioesters with medium chain acyl-CoA dehydrogenase: stereochemistry of inactivation of a flavoenzyme.

The medium chain acyl-CoA dehydrogenase is rapidly inhibited by racemic 3,4-dienoyl-CoA derivatives with a stoichiometry of two molecules of racemate per enzyme flavin. Synthesis of R- and S-3,4-decadienoyl-CoA shows that the R-enantiomer is a potent, stoichiometric, inhibitor of the enzyme. alpha-Proton abstraction yields an enolate to oxidized flavin charge-transfer intermediate prior to adduct formation. The crystal structure of the reduced, inactive enzyme shows a single covalent bond linking the C-4 carbon of the 2,4-dienoyl-CoA moiety and the N5 locus of reduced flavin. The kinetics of reversal of adduct formation by release of the conjugated 2,4-diene were evaluated as a function of both acyl chain length and truncation of the CoA moiety. The adduct is most stable with medium chain length allenic inhibitors. However, the adducts with R-3,4-decadienoyl-pantetheine and -N-acetylcysteamine are some 9- and >100-fold more kinetically stable than the full-length CoA thioester. Crystal structures of these reduced enzyme species, determined to 2.4 A, suggest that the placement of H-bonds to the inhibitor carbonyl oxygen and the positioning of the catalytic base are important determinants of adduct stability. The S-3,4-decadienoyl-CoA is not a significant inhibitor of the medium chain dehydrogenase and does not form a detectable flavin adduct. However, the S-isomer is rapidly isomerized to the trans-trans-2,4-conjugated diene. Protein modeling studies suggest that the S-enantiomer cannot approach close enough to the isoalloxazine ring to form a flavin adduct, but can be facilely reprotonated by the catalytic base. These studies show that truncation of CoA thioesters may allow the design of unexpectedly potent lipophilic inhibitors of fatty acid oxidation.

Carnitine effects on coenzyme A profiles in rat liver with hypoglycin inhibition of multiple dehydrogenases.

To examine the changes in coenzyme A profile and the possible corrective effects of carnitine supplementation in the genetic disorders of mitochondrial beta-oxidation, we carried out experiments using an inhibitor of multiple acyl-CoA dehydrogenase enzymes, methylenecyclopropaneacetic acid (MCPA), in rat hepatocytes. MCPA irreversibly inhibited ketone synthesis from straight-chain fatty acids (butyrate, octanoate, palmitate) and branched-chain fatty acids (alpha-ketoisocaproate) with a parallel 70-90% reduction of hepatocyte acetyl-CoA levels. Alone, MCPA or substrates halved free CoA levels to 15% of total CoA and doubled short- and medium-chain acyl-CoA levels to 30% of total CoA. With MCPA plus substrates combined, free CoA levels were 10% of total CoA, and short- and medium-chain acyl-CoA levels were 45% of total CoA. Comparable changes in CoA profiles were found in a patient with a severe genetic defect in beta-oxidation. Neither the suppression of ketogenesis nor the alterations in CoA profiles induced by MCPA inhibition could be corrected by carnitine supplementation.

Oxidative inactivation of a charge transfer complex in the medium-chain acyl-CoA dehydrogenase.

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 ferricenium 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-thia-octanoyl-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. Synthesis of [2-14C]-3-thia-octanoyl-CoA, followed by isolation of radiolabeled peptide from the modified medium-chain dehydrogenase, showed that inactivation results in labeling the catalytic base, GLU376. Oxidative modification is accompanied by the release of CoASH. A mechanism for inactivation is proposed involving generation of a sulfonium salt which efficiently captures the carboxylate nucleophile.

Recombinant human liver medium-chain acyl-CoA dehydrogenase: purification, characterization, and the mechanism of interactions with functionally diverse C8-CoA molecules.

We offer a large scale purification procedure for the recombinant human liver medium-chain acyl-CoA dehydrogenase (HMCAD). This procedure routinely yield 100-150 mg of homogeneous preparation of the enzyme from 80 L of the Escherichia coli host cells. A comparative investigation of kinetic properties of the human liver and pig kidney enzymes revealed that, except for a few minor differences, both of these enzymes are nearly identical. We undertook detailed kinetic and thermodynamic investigations for the interaction of HMCAD-FAD with three C8-CoA molecules (viz., octanoyl-CoA, 2-octenoyl-CoA, and 2-octynoyl-CoA), which differ with respect to the extent of unsaturation of the alpha-beta carbon center; octanoyl-CoA and 2-octenoyl-CoA serve as the substrate and product of the enzyme, respectively, whereas 2-octynoyl-CoA is known to inactivate the enzyme. Our experimental results demonstrate that all three C8-CoA molecules first interact with HMCAD-FAD to form corresponding Michaelis complexes, followed by two subsequent isomerization reactions. The latter accompany either subtle changes in the electronic structures of the individual components (in case of 2-octenoyl-CoA and 2-octynoyl-CoA ligands), or a near-complete reduction of the enzyme-bound flavin (in case of octanoyl-CoA). The rate and equilibrium constants intrinsic to the above microscopic steps exhibit marked similarity with different C8-CoA molecules. However, the electronic structural changes accompanying the 2-octynoyl-CoA-dependent inactivation of enzyme is 3-4 orders of magnitude slower than the above isomerization reactions. Hence, the octanoyl-CoA-dependent reductive half-reaction and the 2-octynoyl-CoA-dependent covalent modification of the enzyme occur during entirely different microscopic steps. Arguments are presented that the origin of the above difference lies in the protein conformation-dependent orientation of Glu-376 in the vicinity of the C8-CoA binding site.

Medium-chain acyl-CoA dehydrogenase- and enoyl-CoA hydratase-dependent bioactivation of 5,6-dichloro-4-thia-5-hexenoyl-CoA.

5,6-Dichloro-4-thia-5-hexenoic acid (DCTH) is a potent hepato- and nephrotoxin that induces mitochondrial dysfunction in rat liver and kidney. Previous studies indicate that DCTH undergoes fatty acid beta-oxidation-dependent bioactivation. The objectives of the present experiments were to elaborate the bioactivation mechanism of DCTH and to examine the interaction of the coenzyme A thioester of DCTH (DCTH-CoA) with the medium-chain acyl-CoA dehydrogenase. In the presence of the terminal electron acceptor ferricenium hexafluorophosphate (FcPF6), DCTH-CoA was oxidized by the medium-chain actyl-CoA dehydrogenase to give 5,6-dichloro-4-thia-trans-2,5-hexadienoyl-CoA. Enoyl-CoA hydratase catalyzed the conversion of 5,6-dichloro-4-thia-trans-2,5-hexadienoyl-CoA to 5,6-dichloro-4-thia-3-hydroxy-5-hexenoyl-CoA, which eliminated 1,2-dichloroethenethiol and gave malonyl-CoA semialdehyde as a product. Chloroacetic acid was detected as a terminal product derived from 1,2-dichloroethenethiol. Incubation of DCTH-CoA with the medium-chain acyl-CoA dehydrogenase in the absence of FcPF6 gave 3-hydroxypropionyl-CoA as the major product and resulted in the irreversible inactivation of the enzyme. Under these conditions, DCTH-CoA apparently undergoes a beta-elimination reaction to give 1,2-dichloroethenethiol and acryloyl-CoA, which is hydrated to give 3-hydroxypropionyl-CoA as the terminal product. The beta-elimination product 1,2-dichloroethenethiol may yield reactive intermediates that inactivate the dehydrogenase. Enzyme inactivation was rapid, DCTH-CoA concentration-dependent, and blocked by octanoyl-CoA, but not by glutathione. The medium-chain acyl-CoA dehydrogenase was not inactivated by acryloyl-CoA, and little inactivation was observed in the presence of FcPF6. These results show that DCTH-CoA is bioactivated by the mitochondrial fatty acid beta-oxidation system to reactive intermediates. This bioactivation mechanism may account for the observed toxicity of DCTH in vivo and in vitro.

S-2-bromo-acyl-CoA analogues are affinity labels for the medium-chain acyl-CoA dehydrogenase from pig kidney.

S-2-Br-hexanoyl-CoA and the branched chain isomer S-2-Br-4-methyl-pentanoyl-CoA are affinity labels of the medium-chain acyl-CoA dehydrogenase from pig kidney. The straight chain thioester is both a substrate and an irreversible inhibitor of the dehydrogenase. Inactivation of the enzyme is biphasic and is half-complete in 4 min at pH 6.5, 25 degrees C. Although S-2-Br-hexanoyl-CoA can partially reduce the FAD prosthetic group of the dehydrogenase, inactivation results from attachment of one molecular of inhibitor per subunit of the oxidized enzyme. The branched chain analogue is a very weak substrate of the dehydrogenase (0.1% that of octanoyl-CoA), but is almost as effective an inhibitor of the dehydrogenase. Incubation experiments with [14C]S-2-Br-methyl-pentanoyl-CoA followed by the isolation of radiolabeled peptide show that modification of the active site base, GLU376, is responsible for enzyme inactivation. The data are compatible with a simple nucleophilic attack of the carboxylate base on the C-2 atom of these 2-Br-analogues.

Measurement of short-chain acyl-CoA dehydrogenase (SCAD) in cultured skin fibroblasts with hexanoyl-CoA as a competitive inhibitor to eliminate the contribution of medium-chain acyl-CoA dehydrogenase.

Short-chain acyl-CoA dehydrogenase (SCAD) deficiency has so far been reported in only very few patients. This is due, in part, to the problems involved in measuring the activity of SCAD unequivocally. The main reason for this difficulty is that butyryl-CoA, the substrate preferably used for SCAD activity measurements, is also dehydrogenated by medium-chain acyl-CoA dehydrogenase (MCAD). Elimination of this contribution can be achieved by means of immune precipitation with a specific MCAD antibody. We now describe a relatively straightforward assay based on the use of gas chromatography/mass spectrometry for detection. The contribution of MCAD to overall butyryl-CoA dehydrogenation was eliminated by adding excess hexanoyl-CoA to the assay medium. The validity of the method developed was checked by SCAD-activity measurements in fibroblasts from an established SCAD-deficient patient.

3-Methyleneoctanoyl-CoA and 3-methyl-trans-2-octenoyl-CoA: two new mechanism-based inhibitors of medium chain acyl-CoA dehydrogenase from pig kidney.

The medium chain acyl-CoA dehydrogenase catalyzes the FAD-dependent oxidation of a variety of acyl-CoA substrates to the corresponding trans-2-enoyl-CoA thioesters. This work identifies 3-methyleneoctanoyl-CoA and 3-methyl-trans-2-octenoyl-CoA as representatives of a new class of mechanism-based inhibitor of the dehydrogenase. One equivalent of either compound generates an inactive reduced flavin species with low absorption at 450 nm and a shoulder at 320 nm suggestive of an N-5 adduct. Reduction is rapid with the 3-methylene analogue (10/s at 1 degree C), but comparatively slow for 3-methyl-trans-2-octenoyl-CoA (1.1 x 10(-4)/s, under the same conditions). The reduced species is very stable, but the adduct can be slowly displaced with a large excess of octanoyl-CoA. The reduced adduct resists oxidation by the facile one-electron oxidant of the dehydrogenase, ferricenium hexafluorophosphate. Evidence that both isomeric inhibitors generate the same reduced flavin species includes an essentially identical visible spectrum, the same kinetics of displacement using octanoyl-CoA, and the same mixture of products on HPLC after denaturation of the treated enzyme with trichloroacetic acid, methanol, or by boiling. Experiments with the corresponding shorter analogues of these inhibitors, 3-methylenebutanoyl-CoA and 3-methyl-2-butenoyl-CoA confirm and extend these findings. These reduced adducts are less stable, allowing the dehydrogenase to catalyze the interconversion of the unconjugated 3-methylenebutanoyl-CoA to the more stable conjugated 3-methyl-2-butenoyl-CoA thioester (Keq ca. 150). These data suggest that alpha-proton abstraction from the 3-methylene derivatives or gamma-proton removal from the 3-methyl-2-enoyl analogues generates a common carbanionic intermediate which attacks oxidized flavin. As would be expected, the unconjugated 3-methylene derivatives are more effective inhibitors of the dehydrogenase than the thermodynamically more stable 3-methylenoyl analogues.

Inactivation of short-chain acyl-coenzyme A dehydrogenase from pig liver by 2-pentynoyl-coenzyme A.

2-Pentynoyl-CoA is a mechanism-based inactivator of the flavoprotein short-chain acyl-CoA dehydrogenase from pig liver. Inactivation is associated with the formation of an intermediate absorbing at 800 nm and results in the incorporation of 0.86 +/- 0.13 molecules of radiolabeled inhibitor per subunit. A rapid procedure was devised to isolate the labeled peptide. A glutamate residue was identified as the target of 2-pentynoyl-CoA treatment and proved homologous to the proposed catalytic base, GLU376, in the corresponding medium-chain acyl-CoA dehydrogenase sequence. These results are discussed in terms of the lack of conservation of this glutamate residue in the acyl-CoA dehydrogenase enzyme family.

Reductive half-reaction in medium-chain acyl-CoA dehydrogenase: modulation of internal equilibrium by carboxymethylation of a specific methionine residue.

Pig kidney medium-chain acyl-CoA dehydrogenase is specifically alkylated at a methionine residue by treatment with iodoacetate at pH 6.6. This residue corresponds to Met249 in the human medium-chain acyl-CoA dehydrogenase sequence [Kelly, D. P., Kim, J. J., Billadello, J. J., Hainline, B. E., Chu, T. W., & Strauss, A. W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4068-4072]. The S-carboxymethylated dehydrogenase shows a drastically lowered affinity for octanoyl-CoA (from submicromolar to 65 microM), but retains about 23% of the maximal activity of the native enzyme. In addition, alkylation perturbs the internal redox equilibrium: E.FADox.octanoyl-CoA K2 in equilibrium with E.FAD2e.octenoyl-CoA K2 ranges from about 9 for the native enzyme to about 0.2 for the homogeneously modified protein. This effect is not due to a significant change in the redox potential of the free enzyme upon alkylation. Rather, carboxymethylation weakens the preferential binding of enoyl-CoA product to the reduced enzyme (K3) compared to octanoyl-CoA binding to the oxidized dehydrogenase (K1) that is required to pull the substrate thermodynamically uphill. Thus, the ratio of dissociation constants, K1/K3, decreases from about 15,000 for the native enzyme to only 330 upon carboxymethylation of Met249. Binding studies with a variety of acyl-CoA analogues and manipulation of enzyme redox potentials by substitution of the natural prosthetic group by 8-Cl-FAD confirm the thermodynamic effects of alkylation.

Spiropentaneacetic acid as a specific inhibitor of medium-chain acyl-CoA dehydrogenase.

To study the structure-activity relationship between pentanoic acid analogues and the inhibition of fatty acid oxidation, a number of 4-pentenoic and methylenecyclopropaneacetic acid derivatives were prepared. All compounds inhibited palmitoylcarnitine oxidation in rat liver mitochondria, with 50% inhibition occurring at a concentration between 6 and 100 microM. However, only methylenecyclopropaneacetic acid (MCPA) and spiropentaneacetic acid (SPA) showed in vivo inhibitory activity in rats as indicated by the occurrence of dicarboxylic aciduria. Rats treated with SPA excreted metabolites derived only from fatty acid oxidation whereas MCPA-treated rats also excreted metabolites derived from branch-chained amino acid and lysine metabolism. SPA is a specific inhibitor of fatty acid oxidation without affecting amino acid metabolism. The site of inhibition is medium-chain acyl-CoA dehydrogenase (MCAD). In contrast, MCPA inhibited both MCAD and short-chain acyl-CoA dehydrogenase with a stronger inhibition toward the latter. The inhibition of fatty acid oxidation by both inhibitors was partially reversible by glycine or l-carnitine. Since SPA does not form a ring-opened nucleophile such as that proposed for MCPA in the inhibition of FAD prosthetic group in acyl-CoA dehydrogenases, we propose that the irreversible inhibition by SPA occurs by a tight complex without forming a covalent bond to the isoalloxazine ring in FAD.

2-octynoyl coenzyme A is a mechanism-based inhibitor of pig kidney medium-chain acyl coenzyme A dehydrogenase: isolation of the target peptide.

Pig kidney medium-chain acyl-CoA dehydrogenase (EC 1.3.99.3) is irreversibly and stoichiometrically inactivated by [1-14C]-2-octynoyl coenzyme A. The linkage is stable at pH 2-6, but labile under basic conditions. The inhibitor labels a unique tryptic peptide, Ile-Tyr-Gln-Ile-Tyr-Glu-Gly-Thr-Ala-Gln-Ile-Gln-Arg, close to the C-terminus of the protein. The peptide is labeled at Glu-401 with the acyl moiety of the inhibitor but does not contain detectable coenzyme A. Both the inactivation of the dehydrogenase and the appearance of an absorption band at 800 nm show large primary deuterium isotope effects using 4,4'-dideuterio-2-octynoyl-CoA (7.3 and 6.3, respectively). Thus, 2-octynoyl-CoA is a mechanism-based inactivator of the dehydrogenase and is activated by rate-limiting gamma-proton abstraction. Glutamate-401 may be the base that abstracts the pro-R alpha-proton during the dehydrogenation of normal substrates.

Genetic deficiency of short-chain acyl-coenzyme A dehydrogenase in cultured fibroblasts from a patient with muscle carnitine deficiency and severe skeletal muscle weakness.

Genetic deficiency of short-chain acyl-coenzyme A (CoA) dehydrogenase activity was demonstrated in cultured fibroblasts from a 2-yr-old female whose early postnatal life was complicated by poor feeding, emesis, and failure to thrive. She demonstrated progressive skeletal muscle weakness and developmental delay. Her plasma total carnitine level (35 nmol/ml) was low-normal, but was esterified to an abnormal degree (55% vs. control of less than 10%). Her skeletal muscle total carnitine level was low (7.6 nmol/mg protein vs. control of 14 +/- 2 nmol/mg protein) and was 75% esterified. Mild lipid deposition was noted in type I muscle fibers. Fibroblasts from this patient had 50% of control levels of acyl-CoA dehydrogenase activity towards butyryl-CoA as substrate at a concentration of 50 muM in a fluorometric assay based on the reduction of electron transfer flavoprotein. All of this residual activity was inhibited by an antibody against medium-chain acyl-CoA dehydrogenase. These data demonstrated that medium-chain acyl-CoA dehydrogenase accounted for 50% of the activity towards the short-chain substrate, butyryl-CoA, under these conditions, but that antibody against that enzyme could be used to unmask the specific and virtually complete deficiency of short-chain acyl-CoA dehydrogenase in this patient. Fibroblasts from her parents had intermediate levels of activity towards butyryl-CoA, consistent with the autosomal recessive inheritance of this metabolic defect.

Kinetics of suicide substrate:enzyme inactivation. Methylenecyclopropaneacetyl-CoA and general acyl-CoA dehydrogenase.

Kinetics of inactivation of general acyl-CoA dehydrogenase from pig kidney by methylenecyclopropaneacetyl-CoA have been analyzed using the theoretical treatment and exact steady-state kinetic solutions reported by Tatsunami (Tatsunami, S., Yago, N., and Hosoe, M. (1981) Biochim. Biophys. Acta 662, 226-235). Thus practical application of these analytical solutions for an important class of enzyme:substrate reactions has been demonstrated for the first time.

CoA-persulphide: a possible in vivo inhibitor of mammalian short-chain acyl-CoA dehydrogenase.

The characteristic green colour of native short-chain acyl-CoA dehydrogenases (EC 1.3.99.2) results from a charge transfer complex between the FAD prosthetic group and a tightly bound molecule of CoA-persulphide. The native enzyme from ox liver mitochondria was found to have about 60% of its FAD cofactor liganded with CoA-persulphide. When artificially fully liganded with CoA-persulphide, this enzyme was inhibited by 90% in comparison to unliganded enzyme. Enzymic activity could be slowly restored by displacing the CoA-persulphide with high concentrations of butyryl-CoA, the enzyme's physiological substrate. The results show that CoA-persulphide is a potent inhibitor of short-chain acyl-CoA dehydrogenase and may have a physiological role in the regulation of beta-oxidation.

Inactivation of general acyl-CoA dehydrogenase from pig kidney by 2-alkynoyl coenzyme A derivatives: initial aspects.

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.

The suicide inactivation of ox liver short-chain acyl-CoA dehydrogenase by propionyl-CoA. Formation of an FAD adduct.

Propionyl-CoA gave an unexpectedly low turnover (0.03 s-1) and high Km (153 microM) as a substrate for ox liver short-chain acyl-CoA dehydrogenase (SCAD). On addition of an excess of propionyl-CoA to SCAD the flavin A448 decreased to about 30% of its original value and the peak at 368 nm was replaced by one at 335 nm. The decrease in A448 exhibited first-order kinetics and correlated with a first-order decrease in the enzyme's catalytic activity to 22% of the initial value. The flavin, released from propionyl-CoA-treated enzyme with trichloroacetic acid, reacted with O2 to form a stable free radical. This suggests that a reduced N-5 flavin adduct is formed on the enzyme and protected from O2. The released adduct was separated from unmodified flavin and excess propionyl-CoA by h.p.l.c., and was shown by 3H-labelling to contain CoA. The incompleteness of the decrease in the enzyme's A448 and specific activity on incubation with propionyl-CoA probably reflects an equilibrium between covalently and non-covalently bound acyl-CoA, since the spectral changes could be reversed. The enzyme was also re-activated by dilution and incubation with a large molar excess of butyryl-CoA. The rate constant, approx. 2 X 10(-3) s-1, for re-activation, taken with the extrapolated rate constant for the opposing inactivation reaction, 8.9 X 10(-3) s-1, explains the 22% residual activity at equilibrium. The results suggest that propionyl-CoA is a suicide inhibitor for SCAD.

Studies with general acyl-CoA dehydrogenase from pig kidney. Inactivation by a novel type of "suicide" inhibitor, 3,4-pentadienoyl-CoA.

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 10(3) min-1) 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-1) by a large excess of octanoyl-CoA substrate upon reversal of covalent adduct formation. Alternatively, the enzyme inactivator adduct slowly decomposes (t1/2 = 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 alpha-hydrogen as a proton.

Modification of an arginine residue in pig kidney general acyl-coenzyme A dehydrogenase by cyclohexane-1,2-dione.

The flavoenzyme pig kidney general acyl-CoA dehydrogenase (EC 1.3.99.3) is inactivated by cyclohexane-1,2-dione in borate buffer in a reaction that exhibits pseudo-first-order kinetics. Strong protection is afforded by the substrate octanoyl-CoA, as well as by heptadecyl-CoA, a potent competitive inhibitor of the dehydrogenase that does not reduce enzyme flavin. Enzyme exhibiting 10% residual activity in borate buffer contains about 1.3 modified arginine residues per flavin molecule. Very little reduction of the modified enzyme in borate buffer occurs at high concentrations of octanoyl-CoA, in marked contrast with the stoicheiometric reduction of the native enzyme. However, in phosphate buffer alone, the modified enzyme exhibits 55% residual activity and, although binding of substrate is still seriously impaired (apparent Kd=14 microM), excess substrate effects the formation of the characteristic reduced flavin X enoyl-CoA charge-transfer complex. These results suggest that the susceptible arginine residue, though not catalytically essential, is probably within the acyl-CoA-binding site of general acyl-CoA dehydrogenase.