Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.3.1.8 (acyl-CoA dehydrogenase)
785 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The flavoprotein pig kidney general acyl-CoA dehydrogenase contains a single catalytically essential methionine residue/FAD which reacts with iodoacetate at pH 6.6. S-Carboxymethylation of this residue generates an inactive enzyme derivative which retains FAD and the tetrameric structure of the native protein. The derivative binds actanoyl-CoA and palmityol-CoA with concomitant perturbation of the flavin chromophore, but the characterisitic spectrum of the reduced enzyme-enoyl-CoA complex is not observed. In addition, octanyol-CoA strongly protects the native enzyme against alkylation with iodoacetate. These results suggest that the methionine residue is within the active center of acyl-CoA dehydrogenase. Carboxymethylation of this residue may disrupt the precise orientation of the substrate required to achieve transfer of reducing equivalents to the flavin. Pig kidney general acyl-CoA dehydrogenase does not contain exposed catalytically essential cysteine residues.
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PMID:An essential methionine in pig kidney general acyl-CoA dehydrogenase. 745 27

Mitochondrial fatty acid beta-oxidation was studied by incubating stable isotope-labeled fatty acid probes with human fibroblasts in the presence of L-carnitine. The acylcarnitine intermediates produced were analyzed by tandem mass spectrometry. Oxidation by normal fibroblasts produced specific acylcarnitine intermediates corresponding to acyl-CoA dehydrogenase substrates mainly of 10 or less carbons. These probes demonstrated that the pathway, involving all beta-oxidative steps, could be examined. Oxidation of the same precursors by cells with medium chain acyl-CoA dehydrogenase (EC 1.3.99.2) (MCAD) deficiency, which is caused by different DNA mutations, produced acylcarnitine profiles which appear to be specific to this enzyme defect, regardless of the DNA mutation. Increased amounts of octanoyl-, decanoyl-, or decenoylcarnitine were detected. The ratios of octanoylcarnitine to decanoyl- or decenoylcarnitine appear specific for MCAD deficiency. Even though the concentration of labeled decenoylcarnitine (C10:1) was elevated in incubations of MCAD-deficient cells with labeled linoleate or with a fatty acid mixture which included palmitate, oleate, and linoleate, the predominant intermediate was octanoylcarnitines. These results suggest that MCAD-deficient cells readily convert decanoyl-CoA into octanoyl-CoA. This in vitro system could be utilized to study fatty acid oxidation disorders and to study the origins of metabolic intermediates associated with them.
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PMID:Investigation of beta-oxidation intermediates in normal and MCAD-deficient human fibroblasts using tandem mass spectrometry. 755 18

Negative chemical ionization (NCI) mass spectrometry was used to quantify the acyl-CoA intermediates present in human fibroblasts growing in media containing the long-chain fatty acid, palmitate. The acyl-CoA intermediates were detected as the N-acyl pentafluorobenzyl glycinates. In fibroblasts from normal individuals only saturated acyl-CoA esters were detected, supporting the concept that the acyl-CoA dehydrogenase reaction is the rate-limiting step of intramitochondrial fatty acid oxidation. In patients with inherited enzymatic defects of intramitochondrial long-chain fatty acid oxidation, there was not a significant increase in the amount of long-chain acyl-CoA compounds, with palmitoyl-CoA amounts similar to those found in controls. However, there was a sharp decrease in the relative amount of lauroyl-CoA and a resultant sixfold elevation in the palmitoyl-CoA:lauroyl-CoA ratio. In contrast, fibroblasts with a defect involving the transport of fatty acids across the mitochondrial membrane, carnitine palmitoyl transferase 1 deficiency, had a fourfold increase in palmitoyl-CoA. Our results suggest that acyl-CoA esters in biological tissues are readily detectable using NCI mass spectrometry. This approach is significantly more sensitive than previous methods for the detection of these important metabolic intermediates, and may prove useful in the study of fatty acid oxidation in both normal and enzyme-deficient tissues.
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PMID:Long-chain acyl-CoA profiles in cultured fibroblasts from patients with defects in fatty acid oxidation. 755 21

A catalytic intermediate, the so-called "purple complex," of acyl-CoA dehydrogenase is produced on its reaction with the substrate, acyl-CoA. The purple complex is a charge-transfer complex between the reduced enzyme and the product, enoyl-CoA. Resonance Raman spectra of the purple complexes of three acyl-CoA dehydrogenases [short-chain acyl-CoA (SCAD), medium-chain acyl-CoA (MCAD), and isovaleryl-CoA (IVD) dehydrogenases] were measured with excitation at 632.8 nm within charge-transfer absorption bands. The 1,577 cm-1 band of the SCAD purple complex formed in the reaction with butyryl-CoA is mainly associated with the C(1) = O stretching of crotonyl-CoA, judging from the isotopic frequency shifts upon 13C or 18O substitution of butyryl-CoA. The 1,627 cm-1 band of the C(1) = O moiety of crotonyl-CoA in solution shifted downward by 50 cm-1 on complexation with reduced SCAD. This large frequency shift indicates a substantial interaction between C(1) = O and the enzyme, and is further evidence for an appreciable contribution of a polarized form of the C(1) = O moiety in the enzyme-bound enoyl-CoA. This frequency shift can be explained by the hydrogen bond of C(1) = O. The 1,577 cm-1 band of the MCAD purple complex remained constant, regardless of the acyl carbon-chain length (from C4 to C16 of the substrate, acyl-CoA); the alky chain scarcely affected the interaction of the C(1) = O moiety in the active site.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Structural modulation of 2-enoyl-CoA bound to reduced acyl-CoA dehydrogenases: a resonance Raman study of a catalytic intermediate. 759 42

Mitochondrial beta-oxidation involves a family of flavoproteins that introduce a C-C double bond into their fatty acyl-CoA substrates. Deficiencies of these acyl-CoA dehydrogenases lead to fatty acid oxidation disorders involving life-threatening episodes of metabolic derangement. This review focuses on the medium chain acyl-CoA dehydrogenase as the best-understood member of its class. The crystal structure of the enzyme and salient features of its substrate specificity and mechanism of action are summarized. The surprising observation of a catalytically essential amino acid residue that nevertheless is not conserved in the acyl-CoA dehydrogenase family is discussed.
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PMID:Structure and mechanism of action of the acyl-CoA dehydrogenases. 760 36

Medium chain acyl-CoA dehydrogenase from pig kidney catalyzes the oxidation of acyl-CoA thioesters to trans-2-enoyl-CoA derivatives with an optimal chain length of about C-8. The binding energy for alkyl-SCoA thioethers shows no such optimum but increases linearly from C-2 to C-16 with a slope of about 390 cal/-CH2 group. In contrast, four types of CoA-thioester analogues (2-aza-acyl-, 3-thia-acyl-, 3-keto-acyl-, and trans-2-enoyl-) yield an incremental binding energy of about 800 cal/-CH2 group until a chain length of about C-8 is reached. The observed binding energy then decreases, or remains constant, with increasing chain length. Studies with dithiooctanoyl-CoA and 2-azadithiooctanoyl-CoA show that the C = S moiety is accommodated poorly by the medium chain dehydrogenase. A model for chain length discrimination, based on the crystal structure of the enzyme [Kim, J. J. P., Wang, M., & Paschke, R. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7523-7527], is proposed in which hydrogen-bonding interactions between enzyme and thioester carbonyl oxygen atom are maximized at optimal chain lengths. Oversized chains decrease the frequency of effective alignment between enzyme and the C-1 to C-3 region of thioester ligands. Thus the extent of polarization of bound 4-thia-trans-2-enoyl-CoA thioesters decreases sharply with chains longer than C-12.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Role of the carbonyl group in thioester chain length recognition by the medium chain acyl-CoA dehydrogenase. 761 1

In a previous paper, we demonstrated that the reductive half-reaction of medium-chain fatty acyl-CoA dehydrogenase (MCAD), utilizing octanoyl-CoA as physiological substrate, generates two (kinetically distinct) forms of the reduced enzyme (MCAD-FADH2) - octenoyl-CoA charge-transfer complexes [Kumar, N.R., & Srivastava, D.K. (1994) Biochemistry 33, 8833-8841]. We present evidence that octenoyl-CoA dissociates from the second (most stable) charge-transfer complex (referred to as CT2) via two alternative ("facile" and "restricted") pathways. The dissociation of octenoyl-CoA via the facile pathway involves the reversal of the overall reductive half-reaction of the enzyme, generating MCAD-FAD - octanoyl-CoA as the Michaelis complex, followed by dissociation of the latter complex into MCAD-FAD + octanoyl-CoA. Hence, via this pathway, octenoyl-CoA is released from the enzyme site in the form of octanoyl-CoA. In contrast, the restricted pathway involves a direct (albeit slow) dissociation of octenoyl-CoA from CT2 to yield MCAD-FADH2 + octenoyl-CoA. The kinetic profile for the dissociation of octenoyl-CoA via the restricted pathway matches the rate of oxidation of the reduced flavin (within CT2) by O2. This suggests that the oxidase activity of the enzyme remains suppressed as long as the reduced enzyme predominates in the form of the charge-transfer complex(es). The oxidase activity of the enzyme emerges concomitantly with the conversion of CT2 to the MCAD-FADH2 - octenoyl-CoA Michaelis complex. The energetic basis for the dissociation of octenoyl-CoA via the facile and restricted pathways and the mechanism of suppression of the oxidase activity of the enzyme are discussed.
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PMID:Facile and restricted pathways for the dissociation of octenoyl-CoA from the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-FADH2-octenoyl-CoA charge-transfer complex: energetics and mechanism of suppression of the enzyme's oxidase activity. 762 13

Isovaleryl-CoA dehydrogenase (IVD) is a homotetrameric flavoenzyme which catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA. E376 of pig medium chain acyl-CoA dehydrogenase (MCAD), a homologous enzyme, has been identified as the active site catalytic residue. Amino acid sequence alignment shows that A375 is the corresponding residue in human IVD. Using the atomic coordinates determined for MCAD, molecular modeling suggests that E254 is the substituting catalytic residue in IVD. To substantiate the importance of this residue for enzyme function, cDNAs for the wild-type human IVD and E254G, E254D, E254Q, and E254G/A375E mutant IVDs were constructed and cloned into a prokaryotic expression vector. The proteins were synthesized in Escherichia coli and purified, and their properties were examined. The catalytic activity of the recombinant wild-type IVD was the highest in the presence of isovaleryl-CoA, and its UV/visible light spectrum in the presence of isovaleryl-CoA showed quenching of its characteristic absorption in the 445-nm region and appearance of absorption at 600 nm. The E254G and E254Q mutant IVDs had no detectable enzymatic activity, and isovaleryl-CoA did not induce quenching of the absorption in the 445-nm region or the appearance of absorption at 600 nm. The E254D mutant IVD had residual activity for isovaleryl-CoA, and its spectrum was altered compared to that of the wild type. The E254G/A375E mutant IVD exhibited catalytic activity toward isovaleryl-CoA, and its spectrum in the absence or presence of the substrate was similar to that of the wild-type IVD.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Identification of the active site catalytic residue in human isovaleryl-CoA dehydrogenase. 764 Feb 68

The acyl-CoA dehydrogenases (ACDs) are a family of mitochondrial enzymes that oxidize straight chain or branched chain acyl-CoAs in the metabolism of fatty acids or branched chain amino acids. Deficiencies in members of this gene family are important causes of human disease. A cDNA encoding the human precursor for a novel member (gene symbol ACADSB) of the ACD gene family has been isolated and characterized. The open reading frame of 1.3 kb encodes a precursor protein of 431 amino acids, which is processed in vitro to yield a mature protein of 399 amino acids. The cDNA has significant sequence similarity to other members of the acyl-CoA dehydrogenase family, with the greatest homology (38%) to the short chain acyl-CoA dehydrogenase. The cDNA was expressed in eukaryotic (COS) and prokaryotic (Escherichia coli) cells, producing a protein of the expected size, with activity toward the short branched chain acyl-CoA derivatives ((S)-2-methylbutyryl-CoA, isobutyryl-CoA, and 2-methylhexanoyl-CoA), as well as toward the short straight chain acyl-CoAs (butyryl-CoA and hexanoyl-CoA).
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PMID:Isolation and expression of a cDNA encoding the precursor for a novel member (ACADSB) of the acyl-CoA dehydrogenase gene family. 769 50

We undertook a comparative investigation of the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-catalyzed reaction utilizing indole-, furyl-, and 4-(dimethylamino)phenyl-substituted propionyl- and acryloyl-CoAs as potential substrate/product pairs. All these propionyl-CoA derivatives undergo MCAD-catalyzed conversion into their corresponding acryloyl-CoAs via both "dehydrogenase" (in the presence of "organic" electron acceptors) and "oxidase" (buffer-dissolved oxygen serving as the electron acceptor) pathways [Johnson, J. K., Wang, Z. X., & Srivastava, D. K. (1992) Biochemistry 31, 10564-10575]. The steady-state kinetic parameters for the enzyme utilizing these substrates reveal that the KmS (for the CoA substrates) and kcatS for the dehydrogenase reaction are at least an order of magnitude higher than those for the oxidase reaction. As with the CoA substrates, the enzyme catalyzes the conversion of indolepropionyl pantetheine phosphate (IPPP) into indoleacryloyl pantetheine phosphate (IAPP) via these two pathways. However, with IPPP as substrate, the Km (for IPPP) and kcat values of the dehydrogenase and oxidase reactions are the same. These, coupled with the spectral changes of the enzyme-product complexes as well as the binding affinities of the enzyme-substrate/product complexes, lead to the following conclusions: (1) The aromatic/heterocyclic group-containing substrates are converted into their corresponding products via both the dehydrogenase and the oxidase pathways. (2) The 3',5'-ADP moiety of the CoA thioester provides a significant fraction of the total binding energy in stabilizing the enzyme-substrate/product complexes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:"Dehydrogenase" and "oxidase" reactions of medium-chain fatty acyl-CoA dehydrogenase utilizing chromogenic substrates: role of the 3',5'-adenosine diphosphate moiety of the coenzyme A thioester in catalysis. 771 65


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