Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.3.99.3 (acyl-CoA dehydrogenase)
 1,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The binding of substrate/product or transition-state intermediates modifies the properties of medium-chain fatty acyl-CoA dehydrogenase (MCAD) by causing the redox potential to shift positive and the oxygen reactivity to slow by 3000-fold. Two ligands, identified as being the most effective in slowing oxygen reactivity, were 2-azaoctanoyl-CoA and 3-thiaoctanoyl-CoA [Wang, R., & Thorpe, C. (1991) Biochemistry 30, 7895-7901]. We have measured the potential shifts caused by the binding of both ligands to determine which is most similar to the potential shift caused by substrate/product mixture, the assumption being that the best transition-state structural intermediate would give the potential shift most similar to that of substrate/product [Lenn, N.D., Stankovich, M.T., & Liu, H. (1990) Biochemistry 29, 10594-10602]. Both ligands shifted the potential positive, but the shift caused by 2-azaoctanoyl-CoA was 65% that of substrate/product, while 3-thiaoctanoyl-CoA was only 20% of that value. This positive shift is proposed to be caused by a resonance form stabilized by the interaction of the catalytically essential carbonyl of the acyl-CoA with two hydrogen bonds from the enzyme, which induces a partial negative charge on the carbonyl and a partial positive charge on carbon 2 of the ligand and carbon 3 of the substrate/product couple. The X-ray structure shows that carbons 2 and 3 of the substrate/product overlap the diazadiene portion of the flavin ring [Kim, J.-J. P., Wang, M., & Paschke, R. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7523-7527].(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of transition-state analogues on the redox properties of medium-chain acyl-CoA dehydrogenase. 776 14

A highly sensitive and reliable method for assaying acyl-CoA oxidase (EC 1.3.99.3) activity was developed. An acyl-CoA oxidase-dependent [1-14C]palmitoyl-CoA degradation to acetyl-CoA, acid-soluble products, was measured by coupling with the multienzyme complex for fatty acid oxidation from Pseudomonas fragi. The activity, more than 2 pmol/min, could be assessed using this method. The activity was dependent on the coupling enzyme (multienzyme complex), coenzymes such as NAD+ and CoA, and oxygen, and the interference of acyl-CoA dehydrogenases was excluded. The activity in human samples of cultured skin fibroblasts and lymphocytes was compatible with the expected activity calculated from the amount of acyl-CoA oxidase protein estimated by immunoblot analysis. The method which was verified in several experiments can be used for clinical diagnosis of acyl-CoA oxidase deficiency and for determination of activity in samples with a low level of acyl-CoA oxidase.
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PMID:A sensitive assay of acyl-coenzyme A oxidase by coupling with beta-oxidation multienzyme complex. 781 Aug 78

The crystal structure of butyryl-CoA dehydrogenase (BCAD) from Megasphaera elsdenii complexed with acetoacetyl-CoA has been solved at 2.5 A resolution. The enzyme crystallizes in the P422 space group with cell dimensions a = b = 107.76 A and c = 153.67 A. BCAD is a bacterial analog of short chain acyl-CoA dehydrogenase from mammalian mitochondria. Mammalian acyl-CoA dehydrogenases are flavin adenine dinucleotide (FAD)-containing enzymes that catalyze the first step in the beta-oxidation of fatty acids. Although specific for substrate chain lengths, they exhibit high sequence homology. The structure of BCAD was solved by the molecular replacement method using the atomic coordinates of pig liver medium chain acyl-CoA dehydrogenase (MCAD). The structure was refined to an R-factor of 19.3%. The overall polypeptide fold of BCAD is similar to that of MCAD. E367 in BCAD is at the same position and in a similar conformation as the catalytic base in MCAD, E376. The main enzymatic differences between BCAD and MCAD are their substrate specificities and the significant oxygen reactivity exhibited by BCAD but not by MCAD. The substrate binding cavity of BCAD is relatively shallow compared to that of MCAD, as consequences of both a single amino acid insertion and differences in the side chains of the helices that make the binding site. The si-face of the FAD in BCAD is more exposed to solvent than that in MCAD. Therefore solvation can stabilize the superoxide anion and considerably increase the rate of oxidation of reduced flavin in the bacterial enzyme.
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PMID:Three-dimensional structure of butyryl-CoA dehydrogenase from Megasphaera elsdenii. 785 27

The three-dimensional structure of medium-chain acyl-CoA dehydrogenase from pig mitochondria in the native form and that of a complex of the enzyme and a substrate (product) have been solved and refined by x-ray crystallographic methods at 2.4-A resolution to R factors of 0.172 and 0.173, respectively. The overall polypeptide folding and the quaternary structure of the tetramer are essentially unchanged upon binding of the ligand, octanoyl (octenoyl)-CoA. The ligand binds to the enzyme at the rectus (re) face of the FAD in the crevice between the two alpha-helix domains and the beta-sheet domain of the enzyme. The fatty acyl chain of the thioester substrate is buried inside of the polypeptide and the 3'-AMP moiety is close to the surface of the tetrameric enzyme molecule. The alkyl chain displaces the tightly bound water molecules found in the native enzyme and the carbonyl oxygen of the thioester interacts with the ribityl 2'-hydroxyl group of the FAD and the main-chain carbonyl oxygen of Glu-376. The C alpha--C beta of the fatty acyl moiety lies between the flavin and the gamma-carboxylate of Glu-376, supporting the role of Glu-376 as the base that abstracts the alpha proton in the alpha--beta dehydrogenation reaction catalyzed by the enzyme. Trp-166 and Met-165 are located at the sinister (si) side of the flavin ring at the surface of the enzyme, suggesting that they might be involved in the interactions with electron transferring flavoprotein. Lys-304, the prevalent mutation site found in patients with medium-chain acyl-CoA dehydrogenase deficiency, is located approximately 20 A away from the active site of the enzyme.
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PMID:Crystal structures of medium-chain acyl-CoA dehydrogenase from pig liver mitochondria with and without substrate. 835 49

We have shown previously that acetoacetyl-CoA bound to medium-chain acyl-CoA dehydrogenase from pig kidney is transformed into an enolate form, O = C(3)-C(2)H = C(1)-O-, and that the interaction between the C(4a) = N(5) moiety of flavin and the O = C(3)-C(2)H = C(1)-O- moiety of acetoacetyl-CoA is important for the charge-transfer interaction [Nishina, Y. et al. (1992) J. Biochem. 111, 699-706]. In this study, we examined four kinds of acyl-CoA dehydrogenases [short-chain acyl-CoA (SCAD), medium-chain acyl-CoA (MCAD), long-chain acyl-CoA (LCAD), and isovaleryl-CoA (IVD) dehydrogenases] from bovine liver. The Raman spectra of non-labeled and isotopically labeled acetoacetyl-CoA in keto-form revealed that the 1,716-cm-1 and 1,650-cm-1 bands were derived from the C(3) = O and the C(1) = O stretching mode, respectively. In the charge-transfer complexes of acetoacetyl-CoA with the four kinds of dehydrogenases, the resonance Raman (RR) bands corresponding to the C(3) = O and the C(1) = O of acetoacetyl-CoA were observed at around 1,643-1,622 and 1,506-1,476 cm-1, respectively, indicating that acetoacetyl-CoA was transformed into the enolate form as the result of the complexation with the enzymes. Further, in RR spectra with excitation at 632.8 nm, within the charge-transfer band of the complexes of acetoacetyl-CoA with the four acyl-CoA dehydrogenases, both bands associated with the C(4a) = N(5) moiety of oxidized flavin and the O = C(3)-C(2)H = C(1)-O- moiety of acetoacetyl-CoA were enhanced, but the benzene portion of oxidized flavin was not. These results indicate that the substrate activating mechanism is common to all four kinds of dehydrogenases, i.e., the interaction between the C(1) = O of acetoacetyl-CoA and the positively polarized atoms of the enzymes located in close proximity to the oxygen atom of C(1) = O is important, and the C(4a) = N(5) moiety of flavin participates in the interaction. Some kinds of 3-ketoacyl-CoAs were tested instead of acetoacetyl-CoA and essentially similar results were obtained. The positions of the bands derived from the C(1)-O- moiety of 3-ketoacyl-CoAs were different by ca. 30 cm-1 in two groups, i.e., ca. 1,475 cm-1 for SCAD and MCAD and ca. 1,505 cm-1 for LCAD and IVD, that is, RR spectra can classify the four dehydrogenases into two groups.
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PMID:Substrate activating mechanism of short-chain acyl-CoA, medium-chain acyl-CoA, long-chain acyl-CoA, and isovaleryl-CoA dehydrogenases from bovine liver: a resonance Raman study on the 3-ketoacyl-CoA complexes. 874 5

The catalytically essential glutamate residue that initiates catalysis by abstracting the substrate alpha-hydrogen as H+ is located at position 376 (mature MCADH numbering) on loop JK in medium chain acyl-CoA dehydrogenase (MCADH). In long chain acyl-CoA dehydrogenase (LCADH) and isovaleryl-CoA dehydrogenase (IVDH), the corresponding Glu carrying out the same function is placed at position 255 on the adjacent helix G. These glutamates thus act on substrate approaching from two opposite regions at the active center. We have implemented the topology of LCADH in MCADH by carrying out the two mutations Glu376Gly and Thr255Glu. The resulting chimeric enzyme, "medium-/long" chain acyl-CoA dehydrogenase (MLCADH) has approximately 20% of the activity of MCADH and approximately 25% that of LCADH with its best substrates octanoyl-CoA and dodecanoyl-CoA, respectively. MLCADH exhibits an enhanced rate of reoxidation with oxygen, however, with a much narrower substrate chain length specificity that peaks with dodecanoyl-CoA. This is the same maximum as that of LCADH and is thus significantly shifted from that of native MCADH (hexanoyl/octanoyl-CoA). The putative, common ancestor of LCADH and IVDH has two Glu residues, one each at positions 255 and 376. The corresponding MCADH mutant, Thr255Glu (glu/glu-MCADH), is as active as MCADH with octanoyl-CoA; its activity/chain length profile is, however, much narrower. The topology of the Glu as H+ abstracting base seems an important factor in determining chain length specificity and reactivity in acyl-CoA dehydrogenases. The mechanisms underlying these effects are discussed in view of the three-dimensional structure of MLCADH, which is presented in the accompanying paper [Lee et al. (1996) Biochemistry 35, 12412-12420].
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PMID:Medium-long-chain chimeric human Acyl-CoA dehydrogenase: medium-chain enzyme with the active center base arrangement of long-chain Acyl-CoA dehydrogenase. 882 75

Crystal structures of the wild type human medium-chain acyl-CoA dehydrogenase (MCADH) and a double mutant in which its active center base-arrangement has been altered to that of long chain acyl-CoA dehydrogenase (LCADH), Glu376Gly/Thr255Glu, have been determined by X-ray crystallography at 2.75 and 2.4 A resolution, respectively. The catalytic base responsible for the alpha-proton abstraction from the thioester substrate is Glu376 in MCADH, while that in LCADH is Glu255 (MCADH numbering), located over 100 residues away in its primary amino acid sequence. The structures of the mutant complexed with C8-, C12, and C14-CoA have also been determined. The human enzyme structure is essentially the same as that of the pig enzyme. The structure of the mutant is unchanged upon ligand binding except for the conformations of a few side chains in the active site cavity. The substrate with chain length longer than C12 binds to the enzyme in multiple conformations at its omega-end. Glu255 has two conformations, "active" and "resting" forms, with the latter apparently stabilized by forming a hydrogen bond with Glu99. Both the direction in which Glu255 approaches the C alpha atom of the substrate and the distance between the Glu255 carboxylate and the C alpha atom are different from those of Glu376; these factors are responsible for the intrinsic differences in the kinetic properties as well as the substrate specificity. Solvent accessible space at the "midsection" of the active site cavity, where the C alpha-C beta bond of the thioester substrate and the isoalloxazine ring of the FAD are located, is larger in the mutant than in the wild type enzyme, implying greater O2 accessibility in the mutant which might account for the higher oxygen reactivity.
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PMID:Crystal structures of the wild type and the Glu376Gly/Thr255Glu mutant of human medium-chain acyl-CoA dehydrogenase: influence of the location of the catalytic base on substrate specificity. 882 76

Mammalian electron transfer flavoproteins (ETF) are heterodimers containing a single equivalent of flavin adenine dinucleotide (FAD). They function as electron shuttles between primary flavoprotein dehydrogenases involved in mitochondrial fatty acid and amino acid catabolism and the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase. The structure of human ETF solved to 2.1-A resolution reveals that the ETF molecule is comprised of three distinct domains: two domains are contributed by the alpha subunit and the third domain is made up entirely by the beta subunit. The N-terminal portion of the alpha subunit and the majority of the beta subunit have identical polypeptide folds, in the absence of any sequence homology. FAD lies in a cleft between the two subunits, with most of the FAD molecule residing in the C-terminal portion of the alpha subunit. Alignment of all the known sequences for the ETF alpha subunits together with the putative FixB gene product shows that the residues directly involved in FAD binding are conserved. A hydrogen bond is formed between the N5 of the FAD isoalloxazine ring and the hydroxyl side chain of alpha T266, suggesting why the pathogenic mutation, alpha T266M, affects ETF activity in patients with glutaric acidemia type II. Hydrogen bonds between the 4'-hydroxyl of the ribityl chain of FAD and N1 of the isoalloxazine ring, and between alpha H286 and the C2-carbonyl oxygen of the isoalloxazine ring, may play a role in the stabilization of the anionic semiquinone. With the known structure of medium chain acyl-CoA dehydrogenase, we hypothesize a possible structure for docking the two proteins.
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PMID:Three-dimensional structure of human electron transfer flavoprotein to 2.1-A resolution. 896 55

Long-chain-acyl-CoA dehydrogenase (LCADH) has been produced by recombinant techniques from the human cDNA and purified after expression in Escherichia coli. Pig kidney LCADH was purified using an optimized method which also produces apparently pure short-chain-acyl-CoA dehydrogenase (SCADH) and medium-chain-acyl-CoA dehydrogenase (MCADH) in good yields. LCADH from both sources has a maximal turnover rate (Vmax of 650-700 min(-1) at pH 7.6) with the best substrates, which is approximately fivefold higher than reported previously. The human enzyme has an approximately fivefold higher Km compared with the pig kidney enzyme with substrates of chain length from C10 to C18 and a significantly different dependence of Vmax on the chain length. Pig kidney LCADH has a similar Vmax/Km with C10 to C14 substrates as MCADH does with C6 to C10 substrates. Recombinant human LCADH, however, is significantly less efficient (approximately fourfold with C12) than purified pig kidney enzyme. We conclude that human LCADH is either quantitatively less important in beta-oxidation than in the pig, or that post-translational modifications, not present in the recombinant human enzyme, are required to optimize human LCADH activity. Our results demonstrate that LCADH is as important as the other acyl-CoA dehydrogenases in fatty acid oxidation at physiological, mitochondrial pH with optimal substrates of chain length C10-C14. The extent of the LCADH-flavin cofactor reduction observed with most substrates and the rate of the subsequent reoxidation with oxygen are markedly different from those found with human medium chain acyl-CoA dehydrogenase. Both LCADH are inactivated by the substrate analogue 2-octynoyl-CoA, possibly via covalent modification of Glu261, the active-site residue involved in deprotonation of the substrate (alpha)C-H.
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PMID:Characterization of human and pig kidney long-chain-acyl-CoA dehydrogenases and their role in beta-oxidation. 918 95

Since 1911, blood sugars have been measured in newborn infants. Significant neonatal hypoglycemia was first reported in 1937. In 1959, the report of transient symptomatic neonatal hypoglycemia generated worldwide reports. This, along with the ongoing advances in studies of energy metabolism, thermal control and oxygen requirements, led to the first conference on Energy and Carbohydrate Metabolism in the newborn in Tokyo, 1965. Subsequently, a number of hypoglycemia syndromes were discovered. Concurrently, pre-, peri- and neonatal care changed dramatically with the survival or very tiny and very sick newborns. These advances in care made previously derived statistical definitions of hypoglycemia irrelevant. New functional definitions are needed to define abnormal glucose concentrations. Significant hypoglycemia is a continuum of low glucose concentrations of varied duration and severity. Its impact depends upon other risk factors as well. In addition, new hypoglycemic syndromes have appeared. These include deficiencies of blood-brain glucose transporters, the association of hyperinsulinemic hypoglycemia with isoimmune thrombocytopenia and a variety of acyl CoA dehydrogenase deficiencies. Concurrently, carbohydrate disorders in infancy appear to be changing. Neonatal diabetes mellitus, previously transient and benign, now shows a high frequency of recurrence and remaining as a permanent condition. Idiopathic ketotic hypoglycemia of infancy has disappeared in the USA. Familial hyperinsulinemic hypoglycemic syndromes of infancy appear to have a good prognosis, respond to medical intervention and have had their genetic defect localized to a specific gene. Current advances promise reliable bedside techniques to measure central nervous system function, cerebral blood flow, endocrine hormones and receptors as well as glucose transporters and specific genetic defects. These data, when correlated with plasma glucose concentrations and central nervous system function and development, should provide a better understanding of the impact of prolonged and profound hypoglycemia on long-term outcome.
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PMID:Neonatal hypoglycemia 30 years later: does it injure the brain? Historical summary and present challenges. 920 Aug 72


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