Mechanism of action of short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases. Direct evidence for carbanion formation as an intermediate step using enzyme-catalyzed C-2 proton/deuteron exchange in the absence of C-3 exchange

The mechanisms of the initial interactions of three rat liver acyl-CoA dehydrogenases (short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases) and their fatty acyl-CoA substrate were studied using enzyme-catalyzed deuterium exchange. The reaction products were identified and quantitated using mass spectroscopy and 1H-NMR. When fatty acyl-CoA substrates were incubated with catalytic amounts of acyl-CoA dehydrogenase in D2O in the absence of an electron acceptor, a rapid monodeuteration of the substrate occurred to replace one of the prochiral C-2 hydrogens, while no C-3 hydrogens were exchanged with deuterium. The C-2 monodeuteration proceeded to the extent of 80% of the total amount of substrate added at 90 min and almost to completion at 120 min. The pKa values and optimum pD values for the C-2 proton/deuteron exchange reactions were 6.0 and 7.5, respectively, for each of the three acyl-CoA dehydrogenases. The apparent turnover numbers were 3.0, 3.3, and 0.5 s-1 for short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases, respectively. These results provide the first direct evidence for carbanion formation via abstraction of a C-2 hydrogen by a base in the enzyme, as the first step of the catalytic pathway of acyl-CoA dehydrogenation. When the acyl-CoA dehydrogenases were reacted with moderate excesses of acyl-CoA substrates in D2O in the absence of an electron acceptor, maximum bleaching of the FAD absorbance and the appearance of the long wavelength absorbance, attributed to a charge transfer complex, were observed. However, the dehydrogenation products, 2-enoyl-CoAs, were produced either not at all or in an amount which represented only a minor fraction of the amount of the enzyme added, while the substrates in the enzyme-substrate complexes rapidly turned over as indicated by the extensive monodeuteration which concomitantly occurred. Unlike previous hypothesis, these results indicate that the hydride ion transfer from C-3 of the substrate to the enzyme-FAD is not yet complete in the charge-transfer complex. The transfer of the hydride ion to alloxazine N-5 and the release of products are completed only in the presence of electron-transfer flavoprotein or another suitable electron acceptor.     

Medium-chain acyl coenzyme A dehydrogenase from pig kidney has intrinsic enoyl coenzyme A hydratase activity

The flavoprotein medium-chain acyl coenzyme A (acyl-CoA) dehydrogenase from pig kidney exhibits an intrinsic hydratase activity toward crotonyl-CoA yielding L-3-hydroxybutyryl-CoA. The maximal turnover number of about 0.5 min-1 is 500-1000-fold slower than the dehydrogenation of butyryl-CoA using electron-transferring flavoprotein as terminal acceptor. trans-2-Octenoyl- and trans-2-hexadecenoyl-CoA are not hydrated significantly. Hydration is not due to contamination with the short-chain enoyl-CoA hydratase crotonase. Several lines of evidence suggest that hydration and dehydrogenation reactions probably utilize the same active site. These two activities are coordinately inhibited by 2-octynoyl-CoA and (methylenecyclopropyl)acetyl-CoA [whose targets are the protein and flavin adenine dinucleotide (FAD) moieties of the dehydrogenase, respectively]. The hydration of crotonyl-CoA is severely inhibited by octanoyl-CoA, a good substrate of the dehydrogenase. The apoenzyme is inactive as a hydratase but recovers activity on the addition of FAD. Compared with the hydratase activity of the native enzyme, the 8-fluoro-FAD enzyme exhibits a roughly 2-fold increased activity, whereas the 5-deaza-FAD dehydrogenase is only 20% as active. A mechanism for this unanticipated secondary activity of the acyl-CoA dehydrogenase is suggested.     

4-Thia-trans-2-alkenoyl-CoA derivatives: properties and enzymatic reactions

4-Thiaacyl-CoA analogues, in which the 4-methylene group is replaced by a thioether sulfur atom, represent new chromophoric substrates of acyl-CoA dehydrogenases and oxidase. The corresponding 4-thia-trans-2-enoyl-CoA products exhibit a strong new absorption band (extinction coefficient 22 mM-1 cm-1) that is red shifted from 312 to 338 nm upon binding to the medium-chain acyl-CoA dehydrogenase. 4-Thiaoctanoyl-CoA reduces the dehydrogenase several-fold slower than octanoyl-CoA, although in turnover it is dehydrogenated 1.5-fold faster. The redox potential of 4-thia analogues is some 30 mV more negative than that of their unsubstituted counterparts. 4-Thia-trans-2-enoyl-CoA derivatives are slowly hydrated by enoyl-CoA hydratase (EC 4.2.1.17) to the corresponding thiohemiacetal which fragments nonenzymatically to 1 equiv each of malonylsemialdehyde-CoA and alkanethiol. This fragmentation reaction might explain the release of methanethiol during the transamination pathway of methionine degradation. 4-Oxaoctanoyl-CoA is a much poorer substrate and kinetic reductant of acyl-CoA dehydrogenase and oxidase than the 4-thia analogue. The corresponding enoyl-CoA product is also fragmented by the hydratase, yielding butanol and malonylsemialdehyde-CoA. Thus, 4-heterosubstituted acyl-CoA derivatives provide new tools for the study of beta-oxidation enzymes.     

Acyl-CoA oxidase from Candida tropicalis

Acyl coenzyme A oxidase (acyl-CoA oxidase) has been isolated in good yield from Candida tropicalis pK 233 grown on n-alkanes. Gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and measurement of flavin content suggest that the oxidase is an octamer of Mr 75 000 subunits each containing one flavin. The oxidase yields the red semiquinone form on dithionite or photochemical reduction, slowly forms an N-5 adduct with 0.16 M sulfite at pH 7.4, and is rapidly reduced by borohydride, forming the 3,4-dihydroflavin isomer. The red flavosemiquinone is only kinetically stabilized with respect to disproportionation in the free enzyme but is thermodynamically stabilized on binding enoyl-CoA derivatives. The enzyme is reduced by butyryl-, octanoyl-, and palmitoyl-CoA without formation of prominent long-wavelength bands. Acyl-CoA oxidase and the acyl-CoA dehydrogenases share many similarities in their interaction with CoA derivatives. For example, both enzymes stabilize the anionic radical on binding enoyl-CoA derivatives, both dehydrogenate 2-oxoheptadecyldethio-CoA but cannot utilize S-heptadecyl-CoA, both form long-wavelength bands with CoA persulfide species, and both enzymes are attacked by the suicide substrates 3,4-pentadienoyl-CoA and (methylene-cyclopropyl)acetyl-CoA at the flavin prosthetic group.     

Purification and properties of acyl coenzyme A dehydrogenases from bovine liver. Formation of 2-trans,4-cis-decadienoyl coenzyme A

Three straight chain acyl-CoA dehydrogenases were purified to apparent homogeneity from bovine liver using 40-70% (NH4)2SO4 precipitation, gel filtration, DEAE-cellulose column chromatography, and preparative electrophoresis. Separation of the acyl-CoA dehydrogenases by these procedures has been efficiently monitored by two newly developed analytical methods: (i) native staining of acyl-CoA dehydrogenases following separation by electrophoresis in polyacrylamide gels and (ii) determination of general acyl-CoA dehydrogenase by means of a specific substrate, 4-cis-decenoyl-CoA. The three acyl-CoA dehydrogenases were classified into short chain, general, and long chain acyl-CoA dehydrogenases on the basis of their chain length specificities according to the nomenclature proposed by Hall and Kamin (Hall, C. L., and Kamin, H. (1975) J. Biol. Chem. 250, 3470-3486). The enzymes gave single protein bands in polyacrylamide gel electrophoresis under denaturing and nondenaturing conditions, and their subunit and native molecular weights were estimated to be 40,300 and 188,000 for short chain acyl-CoA dehydrogenase, 43,300 and 205,000 for general acyl-CoA dehydrogenase, and 45,200 and 172,000 for long chain acyl-CoA dehydrogenase. Long chain and general acyl-CoA dehydrogenases markedly differed in their substrate specificities toward unsaturated acyl-CoA esters with a double bond at position 4. The former oxidized 4-cis-decenoyl-CoA at a rate of only 2.7% of that obtained with decanoyl-CoA as substrate, while for the latter enzyme 4-cis-decenoyl-CoA was even a slightly better substrate than decanoyl-CoA. 2-trans,4-cis-Decenoyl-CoA was identified as the product of this reaction.     

Interaction of acyl coenzyme A substrates and analogues with pig kidney medium-chain acyl-coA dehydrogenase

Several alkylthio coenzyme A (CoA) derivatives (from ethyl- to hexadecyl-SCoA) have been synthesized to probe the substrate binding site in the flavoprotein medium-chain acyl-CoA dehydrogenase from pig kidney. All bind to apparently equivalent sites with a stoichiometry of four per tetramer. A plot of log Kd vs: hydrocarbon chain length is linear from 2 to 16 carbons with a free energy of binding of 390 cal/methylene group. These data suggest an acyl-binding site of moderate hydrophobicity and imply that the observed substrate specificity of the medium-chain dehydrogenase is not achieved simply by the length of the hydrocarbon binding pocket. Extrapolation of the graph to zero chain length predicts a Kd of 1 mM for the CoA moiety. The difference between this value and the experimentally determined value of 206 microM may be attributed to a contribution from the ionization of the sulfhydryl group in CoASH. The interaction of several eight-carbon intermediates of beta-oxidation (trans-2- and trans-3-octenoyl-CoA and L-3-hydroxy- and 3-ketooctanoyl-CoA) with the dehydrogenase has also been studied. All but the L-3-OH derivative bind tightly to the enzyme (with Kd values in the 50-90 nM range) and are very effective inhibitors of the dehydrogenation of octanoyl-CoA. The trans-3-enoyl analogue produces an immediate, intense, long-wavelength band (lambda max = 820 nm), which probably represents a charge-transfer interaction between the delocalized alpha-carbanion donor and oxidized flavin as the acceptor. The L-3-OH analogue is a reductant of the flavin, yielding 3-ketooctanoyl-CoA.(ABSTRACT TRUNCATED AT 250 WORDS)     

The reductive half-reaction in Acyl-CoA dehydrogenase from pig kidney: studies with thiaoctanoyl-CoA and oxaoctanoyl-CoA analogues

Thia- and oxaoctanoyl-CoA derivatives (substituted at the C-3 and C-4 positions) have been synthesized to prove 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-1 cm-1 at pH 7.6). The intensity of this band increases about 20% from pH 6.0 to pH 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 alpha-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 alpha-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. The corresponding 4-oxa analogue is a much poorer substrate (10% of the rate shown by octanoyl-CoA) but again effects substantially complete reduction of the flavin chromophore in the dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton abstraction reaction, steady-state kinetics, and oxidation-reduction potential of human glutaryl-CoA dehydrogenase

Glutaryl-CoA dehydrogenase catalyzes the oxidation of glutaryl-CoA to crotonyl-CoA and CO(2) in the mitochondrial degradation of lysine, hydroxylysine, and tryptophan. We have characterized the human enzyme that was expressed in Escherichia coli. Anaerobic reduction of the enzyme with sodium dithionite or substrate yields no detectable semiquinone; however, like other acyl-CoA dehydrogenases, the human enzyme stabilizes an anionic semiquinone upon reduction of the complex between the enzyme and 2,3-enoyl-CoA product. The flavin potential of the free enzyme determined by the xanthine-xanthine oxidase method is -0.132 V at pH 7.0, slightly more negative than that of related flavoprotein dehydrogenases. A single equivalent of substrate reduces 26% of the dehydrogenase flavin, suggesting that the redox equilibrium on the enzyme between substrate and product and oxidized and reduced flavin is not as favorable as that observed with other acyl-CoA dehydrogenases. This equilibrium is, however, similar to that observed in isovaleryl-CoA dehydrogenase. Comparison of steady-state kinetic constants of glutaryl-CoA dehydrogenase with glutaryl-CoA and the alternative substrates, pentanoyl-CoA and hexanoyl-CoA, suggests that the gamma-carboxyl group of glutaryl-CoA stabilizes the enzyme-substrate complex by at least 5.7 kJ/mol, perhaps by interaction with Arg94 or Ser98. Glu370 is positioned to function as the catalytic base, and previous studies indicate that the conjugate acid of Glu370 also protonates the transient crotonyl-CoA anion following decarboxylation [Gomes, B., Fendrich, G. , and Abeles, R. H. (1981) Biochemistry 20, 3154-3160]. Glu370Asp and Glu370Gln mutants of glutaryl-CoA dehydrogenase exhibit 7% and 0. 04% residual activity, respectively, with human electron-transfer flavoprotein; these mutations do not grossly affect the flavin redox potentials of the mutant enzymes. The reduced catalytic activities of these mutants can be attributed to reduced extent and rate of substrate deprotonation based on experiments with the nonoxidizable substrate analogue, 3-thiaglutaryl-CoA, and kinetic experiments. Determination of these fundamental properties of the human enzyme will serve as the basis for future studies of the decarboxylation reaction which is unique among the acyl-CoA dehydrogenases.     

Thermodynamic regulation of human short-chain acyl-CoA dehydrogenase by substrate and product binding

Human short-chain acyl-CoA dehydrogenase (hSCAD) catalyzes the first matrix step in the mitochondrial beta-oxidation cycle for substrates with four and six carbons. Previous studies have shown that the act of substrate/product binding induces a large enzyme potential shift in acyl-CoA dehydrogenases. The objective of this work was to examine the thermodynamic regulation of this process through direct characterization of the electrochemical properties of hSCAD using spectroelectrochemical methodology. A large amount of substrate activation was observed in the enzymatic reaction of hSCAD (+33 mV), the greatest magnitude measured in any acyl-CoA dehydrogenase to date. To examine the role of the substrate as well as the product in electron transfer by hSCAD, a catalytic base mutation (E368Q) was constructed. The E368Q mutation inactivates the reductive and oxidative pathways such that the individual effects of substrate and product binding on the redox potential can be investigated. Optimal substrate (butyryl-CoA) was seen to shift the flavin redox potential slightly more positive (+38 mV) than did optimal product (crotonyl-CoA) (+31 mV), a finding opposite of that observed in another short-chain enzyme, bacterial SCAD. These results indicate that substrate redox activation occurs in hSCAD leading to a large enzyme midpoint potential shift. Substrate binding in hSCAD appears to make a larger contribution than does product to thermodynamic modulation.     

Mitochondrial enzymes responsible for oxidizing medium-chain fatty acids in developing rat skeletal muscle, heart, and liver

Prior to weaning, medium-chain fatty acids constitute an important energy source in the developing rat. Fatty acid oxidation rates increase with age in most developing tissues, but the pattern of this increase may vary according to the role of the particular organ. In skeletal muscle, heart, and liver of developing rats, we measured mitochondrial activities of long- and short-chain enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and long- and short-chain acyl-CoA thiolase. In skeletal muscle, the pattern of development in fatty acid oxidation enzymes favored utilization of long-chain rather than medium-chain fatty acids. In liver, enzyme activities for medium-chain fatty acids were highest prior to weaning. Heart occupied a position intermediate between skeletal muscle and liver.     

A novel approach to the characterization of substrate specificity in short/branched chain Acyl-CoA dehydrogenase

Rat and human short/branched chain acyl-CoA dehydrogenases exhibit key differences in substrate specificity despite an overall amino acid identity of 85% between them. Rat short/branched chain acyl-CoA dehydrogenases (SBCAD) are more active toward substrates with longer carbon side chains than human SBCAD, whereas the human enzyme utilizes substrates with longer primary carbon chains. The mechanism underlying this difference in substrate specificity was investigated with a novel surface plasmon resonance assay combined with absorbance and circular dichroism spectroscopy, and kinetics analysis of wild type SBCADs and mutants with altered amino acid residues in the substrate binding pocket. Results show that a relatively few amino acid residues are critical for determining the difference in substrate specificity seen between the human and rat enzymes and that alteration of these residues influences different portions of the enzyme mechanism. Molecular modeling of the SBCAD structure suggests that position 104 at the bottom of the substrate binding pocket is important in determining the length of the primary carbon chain that can be accommodated. Conformational changes caused by alteration of residues at positions 105 and 177 directly affect the rate of electron transfer in the dehydrogenation reactions, and are likely transmitted from the bottom of the substrate binding pocket to beta-sheet 3. Differences between the rat and human enzyme at positions 383, 222, and 220 alter substrate specificity without affecting substrate binding. Modeling predicts that these residues combine to determine the distance between the flavin ring of FAD and the catalytic base, without changing the opening of the substrate binding pocket.     

Acyl-CoA dehydrogenases. A mechanistic overview.

Acyl-CoA dehydrogenases constitute a family of flavoproteins that catalyze the alpha,beta-dehydrogenation of fatty acid acyl-CoA conjugates. While they differ widely in their specificity, they share the same basic chemical mechanism of alpha,beta-dehydrogenation. Medium chain acyl-CoA dehydrogenase is probably the best-studied member of the class and serves as a model for the study of catalytic mechanisms. Based on medium chain acyl-CoA dehydrogenase we discuss the main factors that bring about catalysis, promote specificity and determine the selective transfer of electrons to electron transferring flavoprotein. The mechanism of alpha,beta-dehydrogenation is viewed as a process in which the substrate alphaC-H and betaC-H bonds are ruptured concertedly, the first hydrogen being removed by the active center base Glu376-COO- as an H+, the second being transferred as a hydride to the flavin N(5) position. Hereby the pKa of the substrate alphaC-H is lowered from > 20 to approximately 8 by the effect of specific hydrogen bonds. Concomitantly, the pKa of Glu376-COO- is also raised to 8-9 due to the decrease in polarity brought about by substrate binding. The kinetic sequence of medium chain acyl-CoA dehydrogenase is rather complex and involves several intermediates. A prominent one is the molecular complex of reduced enzyme with the enoyl-CoA product that is characterized by an intense charge transfer absorption and serves as the point of transfer of electrons to the electron transferring flavoprotein. These views are also discussed in the context of the accompanying paper on the three-dimensional properties of acyl-CoA dehydrogenases.     

The reductive half-reaction in acyl-CoA oxidase from Candida tropicalis: interaction with acyl-CoA analogues and an unusual thioesterase activity

A series of acyl-CoA analogues has been used to probe the substrate binding site and reductive half-reaction of acyl-CoA oxidase from the alkane utilizing yeast Candida tropicalis. Alkyl-SCoA thioethers, from octyl- to hexadecyl-SCoA, bind to the oxidase with progressively larger spectral perturbation of the flavin chromophore and with an incremental binding energy of about 260 cal/methylene group. The hydrocarbon binding subsite for acyl-CoA oxidase appears extensive and only weakly hydrophobic. CoA binding per se appears to contribute about 2.8 kcal to the observed binding energy. A number of acyl-CoA analogues such as 3-thia-acyl-, 3-oxa-acyl-, trans-3-enoyl-, and 3-keto-acyl-CoA derivatives form charge transfer complexes with the oxidase, but these long wavelength bands are both less pronounced and much less stable than those encountered with the acyl-CoA dehydrogenases. This instability reflects an intrinsic thioesterase activity of the oxidase which is observed with those ligands forming enolate to oxidized flavin charge-transfer complexes, but not with normal substrates such as palmitoyl-CoA. Chemical precedent suggests that these enzyme-bound enolates eliminate CoA via a ketene intermediate. The differences in behavior between acyl-CoA oxidase and dehydrogenase toward the ligands used in this work are discussed in terms of the need to exclude oxygen from productive encounters with substrate-reduced dehydrogenase.     

Binding, hydration, and decarboxylation of the reaction intermediate glutaconyl-coenzyme A by human glutaryl-CoA dehydrogenase.

Glutaconyl-coenzyme A (CoA) is the presumed enzyme-bound intermediate in the oxidative decarboxylation of glutaryl-CoA that is catalyzed by glutaryl-CoA dehydrogenase. We demonstrated glutaconyl-CoA bound to glutaryl-CoA dehydrogenase after anaerobic reduction of the dehydrogenase with glutaryl-CoA. Glutaryl-CoA dehydrogenase also has intrinsic enoyl-CoA hydratase activity, a property of other members of the acyl-CoA dehydrogenase family. The enzyme rapidly hydrates glutaconyl-CoA at pH 7.6 with a k(cat) of 2.7 s(-1). The k(cat) in the overall oxidation-decarboxylation reaction at pH 7.6 is about 9 s(-1). The binding of glutaconyl-CoA was quantitatively assessed from the K(m) in the hydratase reaction, 3 microM, and the K(i), 1.0 microM, as a competitive inhibitor of the dehydrogenase. These values compare with K(m) and K(i) of 4.0 and 12.9 microM, respectively, for crotonyl-CoA. Glu370 is the general base catalyst in the dehydrogenase that abstracts an alpha-proton of the substrate to initiate the catalytic pathway. The mutant dehydrogenase, Glu370Gln, is inactive in the dehydrogenation and the hydratase reactions. However, this mutant dehydrogenase decarboxylates glutaconyl-CoA to crotonyl-CoA without oxidation-reduction reactions of the dehydrogenase flavin. Addition of glutaconyl-CoA to this mutant dehydrogenase results in a rapid, transient increase in long-wavelength absorbance (lambda(max) approximately 725 nm), and crotonyl-CoA is found as the sole product. We propose that this 725 nm-absorbing species is the delocalized crotonyl-CoA anion that follows decarboxylation and that the decay is the result of slow protonation of the anion in the absence of the general acid catalyst, Glu370(H(+)). In the absence of detectable oxidation-reduction, the data indicate that oxidation-reduction of the dehydrogenase flavin is not essential for decarboxylation of glutaconyl-CoA.     

Assay of Acyl-CoA Dehydrogenase Activity in Frozen Muscle Biopsies: Application to Medium-Chain Acyl-CoA Dehydrogenase Deficiency

The acyl-CoA dehydrogenases (ACDs) are mitochondrial enzymes that dehydrogenate acyl-coenzyme A esters of different chain lengths. Inherited deficiencies of these dehydrogenases are commonly associated with muscle weakness and lipid storage. Numerous assays including spectrophotometric, fluorometric, chemical, and radiochemical procedures have been used, but there is need for a rapid, reproducible assay for the different acyl-CoA dehydrogenases in small frozen samples of human muscle biopsies. We describe a comparative study of dye-linked spectrophotometric assays of the long, medium, and short chain acyl-CoA dehydrogenases in frozen rat and human muscle samples. An optimal procedure is described confirming the value of glass-glass homogenization and assay of a 600g supernatant. Higher activities for all acyl-CoA dehydrogenases, citrate synthase, and cytochrome c oxidase were obtained in rat in contrast to human. The substrate-linked dye reduction method was found superior to the ferricenium or electron transfer flavoprotein acceptor systems. Application of the phenazine ethosulfate-DCPIP-linked method to medium-chain acyl-CoA dehydrogenase (MCAD) was studied in detail and the effect of immunoprecipitation of MCAD allowed for the determination of substrate specificity and the degree of crossover between long-, medium-, and short-chain ACD activity following immunoprecipitation. Finally, a comparison of the specificity and validity of the assay in a patient with MCAD deficiency was performed.     

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.     

An essential cysteine residue located in the vicinity of the FAD-binding site in short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases from rat liver mitochondria

Medium-chain and long-chain acyl-CoA dehydrogenases from rat liver have been purified in two forms, holoenzymes containing FAD and apoenzymes which do not contain this cofactor. In contrast, short-chain acyl-CoA dehydrogenase can only be isolated as the holoenzyme. Marked differences in the reactivity to organic sulfhydryl reagents were observed between the apo and holo forms of these enzymes. While the two apoenzymes were severely inactivated by N-ethylmaleimide (NEM), p-chloromercuribenzoate (pCMB), and iodoacetate (IAA), the two corresponding holoenzymes were not susceptible to these reagents. The inactivation of the two apoenzymes by NEM followed pseudo-first order kinetics. Incubation of the apoenzymes with FAD completely prevented the inactivation by the organic sulfhydryl reagents. Methylmercury halides (iodide or chloride) inactivated both the apo and holo forms of medium-chain and long-chain acyl-CoA dehydrogenases. On the other hand, holo-short-chain acyl-CoA dehydrogenase behaved somewhat differently from the other two holoenzymes in that it was inactivated by pCMB (but not NEM or IAA) following a pseudo-first order process. The titration of the two apoenzymes with [14C]NEM and that of the holo-short-chain acyl-CoA dehydrogenase with [14C]pCMB indicated that all three acyl-CoA dehydrogenases contain a single essential cysteine residue/subunit. In the inactivation of holo-medium-chain and holo-long-chain acyl-CoA dehydrogenases with methylmercury halide, the same essential cysteine residue was modified without perturbing or releasing the enzyme-bound FAD. The inactivations of the three holoenzymes by appropriate organic sulfhydryl reagents were prevented by prior incubation with substrate. These experimental results indicate that the essential cysteine residue is located in the vicinity of the FAD- and substrate-binding sites within the active center of the enzymes. It appears, however, that this cysteine residue does not participate directly in FAD binding.     

Novel inactivation of enoyl-CoA hydratase via beta-elimination of 5, 6-dichloro-7,7,7-trifluoro-4-thia-5-heptenoyl-CoA

5,6-Dichloro-7,7,7-trifluoro-4-thia-5-heptenoyl-CoA (DCTFTH-CoA) is an analogue of a class of cytotoxic 4-thiaacyl-CoA thioesters that can undergo a beta-elimination reaction to form highly unstable thiolate fragments, which yield electrophilic thioketene or thionoacyl halide species. Previous work demonstrated that the medium-chain acyl-CoA dehydrogenase both bioactivates and is inhibited by these CoA thioesters through enzyme-catalyzed beta-elimination of the reactive thiolate moiety [Baker-Malcolm, J. F., Haeffner-Gormley, L., Wang, L., Anders, M. W., and Thorpe, C. (1998) Biochemistry 37, 1383-1393]. This paper shows that DCTFTH-CoA can be directly bioactivated by the enoyl-CoA hydratase (ECH) with the release of 1,2-dichloro-3,3,3-trifluoro-1-propenethiolate and acryloyl-CoA. In the absence of competing exogenous trapping agents, DCTFTH-CoA effects rapid and irreversible loss of hydratase activity. The inactivator is particularly effective at pH 9.0, with a stoichiometry approaching 1 mol of DCTFTH-CoA per enzyme subunit. Modification is associated with a new protein-bound chromophore at 360 nm and an increase in mass of 89 +/- 5 per subunit. Surprisingly, ECH exhibiting less than 2% residual hydratase activity retains essentially 100% beta-eliminase activity and continues to generate reactive thiolate species from DCTFTH-CoA. This leads to progressive derivatization of the enzyme with additional UV absorbance, covalent cross-linking of subunits, and an eventual complete loss of beta-eliminase activity. A range of exogenous trapping agents, including small thiol nucleophiles, various proteins, and even phospholipid bilayers, exert strong protection against modification of ECH. Peptide mapping, thiol titrations, UV-vis spectrophotometry, and mass spectrometry show that inactivation involves the covalent modification of Cys62 and/or Cys111 of the recombinant rat liver ECH. These data suggest that enoyl-CoA hydratase is an important enzyme in the bioactivation of DCTFTH-CoA, in a pathway which does not require involvement of the medium-chain acyl-CoA dehydrogenase.     

Resonance Raman study of flavins and the flavoprotein fatty acyl coenzyme A dehydrogenase.

The resonance Raman (RR) spectra of FMN, FAD, FAD in D2O, and 7,8-dimethyl-1, 10-ethyleneisoalloxazinium perchlorate have been obtained by employing KI as a collisional fluorescence-quenching agent. The spectra are very similar to those obtained recently by using the CARS technique to eliminate fluorescence. Spectra have also been obtained for several species in which flavin is known to fluoresce only weakly. We report RR spectra of protonated FMN, FMN semiquinone cation, the general fatty acyl-CoA dehydrogenase, and two "charge-transfer" complexes of fatty acyl-CoA dehydrogenase. Tentative assignment of several vibrational bands can be made on the basis of our flavin spectra. RR spectra of fatty acyl-CoA and its complexes are consistent with the previous hypothesis that visible spectral shifts observed during formation of acetoacetyl-CoA and crotonyl-CoA complexes of fatty acyl-CoA dehydrogenase result from charge-transfer interactions in which the ground state is essentially nonbonding as opposed to interactions in which complete electron transfer occurs to form FAD semiquinone. The only significant change in the RR spectrum of FAD on binding to enzyme occurs in the 1250-cm-1 region of the spectrum, a region associated with delta N--H of N-3. The position of this band in fatty acyl-CoA dehydrogenase and the other flavoproteins studied to date is discussed in terms of hydrogen bonding between flavin and protein.     

Quantitation of the efflux of acylcarnitines from rat heart, brain, and liver mitochondria

The efflux of individual short-chain and medium-chain acylcarnitines from rat liver, heart, and brain mitochondria metabolizing several substrates has been measured. The acylcarnitine efflux profiles depend on the substrate, the source of mitochondria, and the incubation conditions. The largest amount of any acylcarnitine effluxing per mg of protein was acetylcarnitine produced by heart mitochondria from pyruvate. This efflux of acetylcarnitine from heart mitochondria is almost 5 times greater with 1 mM than 0.2 mM carnitine. Apparently the acetyl-CoA generated from pyruvate by pyruvate dehydrogenase is very accessible to carnitine acetyltransferase. Very little acetylcarnitine effluxes from heart mitochondria when octanoate is the substrate except in the presence of malonate. Acetylcarnitine production from some substrates peaks and then declines, indicating uptake and utilization. The unequivocal demonstration that considerable amounts of propionylcarnitine or isobutyrylcarnitine efflux from heart mitochondria metabolizing alpha-ketoisovalerate and alpha-keto-beta-methylvalerate provides evidence for a role (via removal of non-metabolizable propionyl-CoA or slowly metabolizable acyl-CoAs) for carnitine in tissues which have limited capacity to metabolize propionyl-CoA. These results also show propionyl-CoA must be formed during the metabolism of alpha-ketoisovalerate and that extra-mitochondrial free carnitine rapidly interacts with matrix short-chain aliphatic acyl-CoA generated from alpha-keto acids of branched-chain amino acids and pyruvate in the presence and absence of malate.