Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Pigeon liver fatty acid synthetase was inactivated by stoichiometric concentrations of diethylpyrocarbonate (DEP). The second order rate constant for the loss of synthetase activity was similar to the value for enoyl-CoA reductase indicating that ethoxyformylation destroys the ability of the enzyme to reduce the unsaturated acyl intermediate, without significant effect on beta-ketoacyl reductase activity. NADPH provided protection to the enzyme against inactivation by DEP indicating that essential histidine residues are present at the active site. DEP-modified enzyme showed a characteristic absorption maxima at 240 nm confirming the formation of ethoxyformic histidine. The reversal of inactivation by hydroxylamine and a pKa value of 7.0 obtained from the pH-rate profile for inactivation again confirmed the specificity of DEP for histidine. Stoichiometric results showed that two moles of histidine residue per mole of enzyme are essential for the activity of FAS.
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PMID:Evidence for the essential histidine at the NADPH binding site of enoyl-CoA reductase domain of pigeon liver fatty acid synthetase. 920 89

Surfactin synthetase is the enzyme responsible for biosynthesis of the lipoheptapeptide antibiotic surfactin by Bacillus subtilis. Fragments of SrfB1, the L-valine-activating module of the second subunit of surfactin synthetase, were overproduced in Escherichia coli. In addition to a 143-kDa SrfB1 fragment that contains four domains putatively involved in activation (adenylation domain), autoaminoacylation (peptidyl carrier protein (PCP) domain), and peptide bond formation (two condensation domains), subfragments comprising two domains (104-kDa condensation-adenylation and 73-kDa adenylation-PCP), and one domain (18-kDa PCP) were also overproduced in and purified from E. coli as N-terminal hexahistidine fusion proteins. Incubation of these domains with pure Sfp, a phosphopantetheinyl transferase (PPTase) from B. subtilis, and CoA allowed quantitation of posttranslational phosphopantetheinylation of Ser999 by mass spectrometry for the 18-kDa PCP fragment and by radioassay using cosubstrate [3H] pantetheinyl-coenzyme A for all PCP-containing constructs. The phosphopantetheine stoichiometry correlated with the subsequent mole fractions of [14C] valyl groups that could be covalently transferred to these holo-PCP domains. In turn, the catalytic efficiency of intramolecular aminoacylation of the 143-kDa fragment could be compared with the reaction "in trans" between adenylation and PCP fragments of SrfB1. The corresponding holo-PCP domain of the next module, SrfB2, was not detectably aminoacylated by SrfB1, indicative of protein-protein recognition between adenylation and cognate PCP domains. These results should permit future exploration of the timing and specificity of peptide bond formation by this class of biosynthetic enzymes.
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PMID:Stoichiometry and specificity of in vitro phosphopantetheinylation and aminoacylation of the valine-activating module of surfactin synthetase. 948 28

Isovaleryl-CoA dehydrogenase (IVD) is a homotetrameric mitochondrial flavoenzyme which catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA. PCR of IVD genomic and complementary DNA was used to identify mutations occurring in patients with deficiencies in IVD activity. Western blotting, in vitro mitochondrial import, prokaryotic expression, and kinetic studies of IVD mutants were conducted to characterize the molecular defects caused by the amino acid replacements. Mutations leading to Arg21Pro, Asp40Asn, Ala282Val, Cys328Arg, Val342Ala, Arg363Cys, and Arg382Leu replacements were identified. Western blotting of fibroblast extracts and/or in vitro mitochondrial import experiments indicate that the seven precursor IVD mutant peptides, and a previously identified IVD Leu13Pro mutant, are synthesized and imported into mitochondria. While the IVD Leu13Pro, Arg21Pro, and Cys328Arg mutant peptides are rapidly degraded following mitochondrial import, the other mutant peptides exhibit greater mitochondrial stability, though less than the wild-type enzyme. Active IVD Ala282Val, Val342Ala, Arg363Cys, and Arg382Leu mutants were less stable than wild type when produced in Escherichia coli. The Km values of purified IVD Ala282Val, Val342Ala, and Arg382Leu mutants are 27.0, 2. 8, and 6.9 microM isovaleryl-CoA, respectively, compared to 3.1 microM for the wild type, using the electron-transfer flavoprotein (ETF) fluorescence quenching assay. The catalytic efficiency per mole of FAD content of these three mutants is 4.8, 17.0, and 17.0 microM-1*min-1, respectively, compared to 170 microM-1*min-1 for wild type.
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PMID:Characterization of molecular defects in isovaleryl-CoA dehydrogenase in patients with isovaleric acidemia. 966 41

The recent discovery that sterol carrier protein-2 (SCP-2) binds long chain++ (LCFA-CoA) with high affinity (A. Frolov et al., J. Biol. Chem. 271 (1997) 31878-31884) suggests new possible functions of this protein in LCFA-CoA metabolism. The purpose of the present investigation was to determine whether SCP-2 differentially modulated microsomal LCFA-CoA transacylation to cholesteryl esters, triacylglycerols, and phospholipids in vitro. Microsomal acyl-CoA:cholesterol acyltransferase (ACAT) activity measured with liposomal membrane cholesterol donors depended on substrate LCFA-CoA level, mol% cholesterol in the liposomal membrane, and total amount of liposomal cholesterol. As compared to basal activity without liposomes, microsomal ACAT was inhibited 30-50% in the presence of cholesterol poor (1.4 mol%) liposomes. In contrast, cholesterol rich (>25 mol%) liposomes stimulated ACAT up to 6.4-fold compared to basal activity without liposomes and nearly 10-fold as compared to cholesterol pool (1.4 mol%) liposomes. Increasing oleoyl-CoA reversed the inhibition of microsomal ACAT by cholesterol poor (1.4 mol%) liposomes, but did not further stimulate ACAT in the presence of cholesterol rich (35 mol%) liposomes. In contrast, high (100 microM) oleoyl-CoA inhibited ACAT nearly 3-fold. This inhibition was reversed by LCFA-CoA binding proteins, bovine serum albumin (BSA) and SCP-2. SCP-2 was 10-fold more effective (mole for mole) than BSA in reversing LCFA-CoA inhibited microsomal ACAT. Concomitantly, under conditions in which SCP-2 stimulated ACAT it equally enhanced transacylation of oleoyl-CoA into phospholipids, and 5.2-fold enhanced oleoyl-CoA transacylation to triacylglycerols. In summary, SCP-2 appeared to exert its greatest effects on microsomal transacylation in vitro by reversing LCFA-CoA inhibition of ACAT and by differentially targeting LCFA-CoA to triacylglycerols. These data suggest that the high affinity interaction of SCP-2s with LCFA-CoA may be physiologically important in microsomal transacylation reactions.
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PMID:Microsomal long chain fatty acyl-CoA transacylation: differential effect of sterol carrier protein-2. 1049 8

Pigeon liver fatty acid synthetase (FAS) was inactivated irreversibly by stoichiometric concentration of o-phthalaldehyde exhibiting a bimolecular kinetic process. FAS-o-phthalaldehyde adduct gave a characteristic absorption maxima at 337 nm. Moreover this derivative showed fluorescence emission maxima at 412 nm when excited at 337 nm. These results were consistent with isoindole ring formation in which the -SH group of cysteine and epsilon-NH2 group of lysine participate in the reaction. The inactivation is caused by the reaction of the phosphopantetheine -SH group since it is protected by either acetyl- or malonyl-CoA. The enzyme incubated with iodoacetamide followed by o-phthalaldehyde showed no change in fluorescence intensity but decrease in intensity was found in the treatment of 2,4,6-trinitrobenzenesulphonic acid (TNBS), a lysine specific reagent with the enzyme prior to o-phthalaldehyde addition. As o-phthalaldehyde did not inhibit enoyl-CoA reductase activity, so nonessential lysine is involved in the o-phthalaldehyde reaction. Double inhibition experiments showed that 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), a thiol specific reagent, binds to the same cysteine which is also involved in the o-phthalaldehyde reaction. Stoichiometric results indicated that 2 moles of o-phthalaldehyde were incorporated per mole of enzyme molecule upon complete inactivation.
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PMID:Nature of o-phthalaldehyde reaction with pigeon liver fatty acid synthetase. 1054 64

Recombinantly expressed human ATP:citrate lyase was purified from E. coli, and its kinetic behavior was characterized before and after phosphorylation. Cyclic AMP-dependent protein kinase catalyzed the incorporation of only 1 mol of phosphate per mole of enzyme homotetramer, and glycogen synthase kinase-3 incorporated an additional 2 mol of phosphate into the phosphorylated protein. Isoelectric focusing revealed that all of the phosphates were incorporated into only one of the four enzyme subunits. Phosphorylation resulted in a 6-fold increase in V(max) and the conversion of citrate dependence from sigmoidal, displaying negative cooperativity, to hyperbolic. The phosphorylated recombinant enzyme is more similar to the enzyme isolated from mammalian tissues than unphosphorylated enzyme with respect to the K(m) for citrate, CoA, and ATP, and the specific activity. Fructose 6-phosphate was found to be a potent activator (60-fold) of the unphosphorylated recombinant enzyme, with half-maximal activation at 0.16 mM, which results in a decrease in the apparent K(m) for citrate and ATP, as well as an increase in the V(max) of the reaction. Thus, human ATP:citrate lyase activity is regulated in vitro allosterically by phosphorylated sugars as well as covalently by phosphorylation.
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PMID:Phosphorylation of recombinant human ATP:citrate lyase by cAMP-dependent protein kinase abolishes homotropic allosteric regulation of the enzyme by citrate and increases the enzyme activity. Allosteric activation of ATP:citrate lyase by phosphorylated sugars. 1065 65

2,4-Dienoyl-CoA reductase is an enzyme that is required for the beta-oxidation of unsaturated fatty acids with even-numbered double bonds. The 2,4-dienoyl-CoA reductase from Escherichia coli was studied to explore the catalytic and structural properties that distinguish this enzyme from the corresponding eukaryotic reductases. The E. coli reductase was found to contain 1 mol of flavin mononucleotide and 4 mol each of acid-labile iron and sulfur in addition to 1 mol of flavin adenine dinucleotide per mole of protein. Redox titrations revealed a requirement for 5 mol of electrons to completely reduce 1 mol of enzyme and provided evidence for the formation of a red semiquinone intermediate. The reductase caused a significant polarization of the substrate carbonyl group as indicated by an enzyme-induced red shift of 38 nm in the spectrum of 5-phenyl-2,4-pentadienoyl-CoA. However, suspected cis --> trans isomerase and Delta(3),Delta(2)-enoyl-CoA isomerase activities were not detected in this enzyme. It is concluded that the 2, 4-dienoyl-CoA reductases from E. coli and eukaryotic organisms are structurally and mechanistically unrelated enzymes that catalyze the same type of reaction with similar efficiencies.
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PMID:2,4-Dienoyl-CoA reductase from Escherichia coli is a novel iron-sulfur flavoprotein that functions in fatty acid beta-oxidation. 1093 94

The goal of this study was to examine arachidonic acid (AA) metabolism by murine bone marrow-derived mast cells (BMMC) during apoptosis induced by cytokine depletion. BMMC deprived of cytokines for 12-48 h displayed apoptotic characteristics. During apoptosis, levels of AA, but not other unsaturated fatty acids, correlated with the percentage of apoptotic cells. A decrease in both cytosolic phospholipase A(2) expression and activity indicated that cytosolic phospholipase A(2) did not account for AA mobilization during apoptosis. Free AA accumulation is also unlikely to be due to decreases in 5-lipoxygenase and/or cyclooxygenase activities, since BMMC undergoing apoptosis produced similar amounts of leukotriene B(4) and significantly greater amounts of PGD(2) than control cells. Arachidonoyl-CoA synthetase and CoA-dependent transferase activities responsible for incorporating AA into phospholipids were not altered during apoptosis. However, there was an increase in arachidonate in phosphatidylcholine (PC) and neutral lipids concomitant with a 40.7 +/- 8.1% decrease in arachidonate content in phosphatidylethanolamine (PE), suggesting a diminished capacity of mast cells to remodel arachidonate from PC to PE pools. Further evidence of a decrease in AA remodeling was shown by a significant decrease in microsomal CoA-independent transacylase activity. Levels of lyso-PC and lyso-PE were not altered in cells undergoing apoptosis, suggesting that the accumulation of lysophospholipids did not account for the decrease in CoA-independent transacylase activity or the induction of apoptosis. Together, these data suggest that the mole quantities of free AA closely correlated with apoptosis and that the accumulation of AA in BMMC during apoptosis was mediated by a decreased capacity of these cells to remodel AA from PC to PE.
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PMID:A decrease in remodeling accounts for the accumulation of arachidonic acid in murine mast cells undergoing apoptosis. 1102 38

A novel multivariable control strategy is developed for alcohol (ethanol and n-pentanol) concentrations in the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(HB-co-HV), a biodegradable polymer by Paracoccus denitrificans ATCC 1774. This controller, which is developed to control the mole fraction of P(HB-co-HV), consists of two parts: one is for ethanol concentration control and the other is for mole fraction control, based on the concept of metabolic flux distribution control. A simple metabolic reaction (MR) model is constructed for flux distribution analysis. The relationship between mole ratio of specific consumption rate of the two alcohols (ethanol and n-pentanol) and the mole fraction of 3HV units in the polymer is linear. This result suggests that the split ratio at a branched point of 3-ketovaleryl-CoA in the P(HB-co-HV) synthetic pathway is constant for several fermentation conditions. When the mole fraction of 3HV units has a target value, the feed rate of n-pentanol becomes a function of the feed rate of ethanol and the set value of 3HV, based on the MR model. The mole fraction of 3HV units successfully reached the target value using this strategy. The mole fraction control strategy is combined with an optimal production strategy based on the optimal profile of the specific growth rate. The combined strategy is realized using multivariable controllers and P(3HB-co-3HV) production is maximized with a given value of mole fraction of 3HV units at the final step of fermentation.
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PMID:Multivariable control of alcohol concentrations in the production of polyhydroxyalkanoates (PHAs) by Paracoccus denitrificans. 1137

Isovaleryl-CoA dehydrogenase (IVD) is a flavoenzyme, which catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA in the leucine catabolism pathway and transfers electrons to the electron-transferring flavoprotein (ETF). IVDs from human and rat have been identified and characterized previously. In this study, the gene coding for Caenorhabditis elegans IVD has been identified from a published cDNA sequence and molecular modeling has been performed using the human IVD atomic coordinates. The coding sequence for the mature form of the enzyme was expressed in Escherichia coli, and the recombinant nematode IVD enzyme was purified to essential homogeneity. Its spectrum is typical of recombinant FAD-containing acyl-CoA dehydrogenases and shows a minor broad absorption band at 650-700 nm characteristic of an IVD:CoA persulfide charge-transfer complex. Following treatment of the enzyme with sodium dithionite to remove the bound CoA persulfide, the K(m) values for isovaleryl-, butyryl-, valeryl-, and hexanoyl-CoA were estimated to be 2.5, 36.2, 10.5, and 33.8 microM, respectively, using the ETF fluorescence reduction assay. The catalytic efficiency (k(cat)/K(m)) for these substrates was 56.9, 1.3, 13.7, and 3.2 microM(-1). min(-1) per mole of FAD, respectively. The apparent binding constant (K(D app)) of the recombinant IVD determined spectrally for isovaleryl-CoA was 0.34 microM. These kinetic parameters confirm that isovaleryl-CoA is the preferred substrate for the purified enzyme. The variability in the protein structure among known and putative IVDs from various species is discussed in the context of possible mechanisms for modulating enzyme activity.
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PMID:Identification of Caenorhabditis elegans isovaleryl-CoA dehydrogenase and structural comparison with other acyl-CoA dehydrogenases. 1138 48


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