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:6.4.1.2 (acetyl-CoA carboxylase)
2,876 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report the molecular cloning and DNA sequence of the gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase. The biotin carboxylase gene encodes a protein of 449 residues that is strikingly similar to amino-terminal segments of two biotin-dependent carboxylase proteins, yeast pyruvate carboxylase and the alpha-subunit of rat propionyl-CoA carboxylase. The deduced biotin carboxylase sequence contains a consensus ATP binding site and a cysteine-containing sequence preserved in all sequenced bicarbonate-dependent biotin carboxylases that may play a key catalytic role. The gene encoding the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase is located upstream of the biotin carboxylase gene and the two genes are cotranscribed. As previously reported by others, the BCCP sequence encoded a protein of 16,688 molecular mass. However, this value is much smaller than that (22,500 daltons) obtained by analysis of the protein. Amino-terminal amino acid sequencing of the purified BCCP protein confirmed the deduced amino acid sequence indicating that BCCP is a protein of atypical physical properties. Northern and primer extension analyses demonstrate that BCCP and biotin carboxylase are transcribed as a single mRNA species that contains an unusually long untranslated leader preceding the BCCP gene. We have also determined the mutational alteration in a previously isolated acetyl-CoA carboxylase (fabE) mutant and show the lesion maps within the BCCP gene and results in a BCCP species defective in acceptance of biotin. Translational fusions of the carboxyl-terminal 110 or 84 (but not 76) amino acids of BCCP to beta-galactosidase resulted in biotinated beta-galactosidase molecules and production of one such fusion was shown to result in derepression of the biotin biosynthetic operon.
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PMID:The gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase. 137 Apr 69

We have studied the apo (unbiotinylated) and holo (biotinylated) forms of BCCP87, an 87-residue COOH-terminal peptide comprising the biotin carrier domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase. The apo protein spontaneously formed disulfide-linked dimers and was modified readily by sulfhydryl reagents, whereas the holo protein remained monomeric and was unreactive toward sulfhydryl reagents unless a protein denaturant was present. These data indicated that the single cysteine residue of the domain (Cys-116) was much more reactive in the apo form of the protein. Incubation of apoBCCP87 with biotin ligase for different times, followed by reaction with fluorescein-5-maleimide, clearly showed that the loss of Cys-116 reactivity was the result of modification with biotin. In addition, reaction of Cys-116 with 5,5'-dithiobis(2-nitrobenzoic acid) showed that apoBCCP87 denatured at lower urea concentrations than holoBCCP87. We also found that apoBCCP87 was at least 10-fold more sensitive than the holo form to proteolysis by a range of proteases. Identification of the cleavage sites indicated that the differences in protease sensitivity could not be attributed to shielding of susceptible bonds by the biotin moiety of the holo protein. These data indicate that a conformational change accompanies biotinylation of the biotin domain. Thus, modification of a beta-turn protruding from the protein surface results in alteration of the overall structure of this protein domain.
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PMID:Covalent modification of an exposed surface turn alters the global conformation of the biotin carrier domain of Escherichia coli acetyl-CoA carboxylase. 932 38

Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase protein, a biotin carboxyl carrier protein, and a carboxyltransferase protein. In this report the overexpression of the genes for the carboxyltransferase component is described. The steady-state kinetics of the recombinant carboxyltransferase are characterized in the reverse direction, in which malonyl-CoA reacts with biocytin to form acetyl-CoA and carboxybiocytin. The initial velocity patterns indicated that the kinetic mechanism is equilibrium-ordered with malonyl-CoA binding before biocytin and the binding of malonyl-CoA to carboxyltransferase at equilibrium. The biotin analogs, desthiobiotin and 2-imidazolidone, inhibited carboxyltransferase. Both analogs exhibited parabolic noncompetitive inhibition, which means that two molecules of inhibitor bind to the enzyme. The pH dependence for both the maximum velocity (V) and the (V/K)biocytin parameters decreased at low pH. A single ionizing group on the enzyme with a pK of 6.2 or lower in the (V/K)biocytin profile and 7. 5 in the V profile must be unprotonated for catalysis. Carboxyltransferase was inactivated by N-ethylmaleimide, whereas malonyl-CoA protected against inactivation. This suggests that a thiol in or near the active site is needed for catalysis. The rate of inactivation of carboxyltransferase by N-ethylmaleimide decreased with decreasing pH and indicated that the pK of the sulfhydryl group had a pK value of 7.3. It is proposed that the thiolate ion of a cysteine acts as a catalytic base to remove the N1' proton of biocytin.
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PMID:Overexpression and kinetic characterization of the carboxyltransferase component of acetyl-CoA carboxylase. 966 99

Plastidic acetyl-CoA carboxylase (ACCase; EC 6.4.1.2), which catalyses the synthesis of malonyl-CoA and is the regulatory enzyme of fatty acid synthesis, is activated by light, presumably under redox regulation. To obtain evidence of redox regulation in vivo, the activity of ACCase was examined in pea chloroplasts isolated from plants kept in darkness (dark-ACCase) or after exposure to light for 1 h (light-ACCase) in the presence or absence of a thiol-reducing agent, dithiothreitol (DTT). The protein level was similar for light-ACCase and dark-ACCase, but the activity of light-ACCase in the absence of DTT was approx. 3-fold that of dark-ACCase. The light-ACCase and dark-ACCase were activated approx. 2-fold and 6-fold by DTT respectively, indicating that light-ACCase was in a much more reduced, active form than the dark-ACCase. This is the first demonstration of the light-dependent reduction of ACCase in vivo. Measurement of the activities of ACCase, carboxyltransferase and biotin carboxylase in the presence and absence of DTT, and the thiol-oxidizing agent, 5, 5'-dithiobis-(2-nitrobenzoic) acid, revealed that the carboxyltransferase reaction, but not the biotin carboxylase reaction, was redox-regulated. The cysteine residue(s) responsible for redox regulation probably reside on the carboxyltransferase component. Measurement of the pH dependence of biotin carboxylase and carboxyltransferase activities in the ACCase suggested that both components affect the activity of ACCase in vivo at a physiological pH range. These results suggest that the activation of ACCase by light is caused partly by the pH-dependent activation of two components and by the reductive activation of carboxyltransferase.
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PMID:Light-dependent changes in redox status of the plastidic acetyl-CoA carboxylase and its regulatory component. 1021 91

Biotin carboxylase from Escherichia coli catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl-CoA carboxylase, which catalyzes the committed step in long-chain fatty acid synthesis. For the carboxylation of biotin to occur, biotin must be deprotonated at its N1' position. Kinetic investigations, including solvent isotope effects and enzyme inactivation by N-ethylmaleimide, suggested a catalytic role for a cysteine residue and led to the proposal of a mechanism for the deprotonation of biotin. The proposed pathway suggests a catalytic base removes a proton from a nearby cysteine residue, forming a thiolate anion, which then abstracts the proton from biotin. Inactivation studies of pyruvate carboxylase, which has an analogous mode of action to biotin carboxylase, suggests the catalytic base in this reaction is a lysine residue. Using the crystal structure of biotin carboxylase, cysteine 230 and lysine 238 were identified as the likely active-site residues that act as this acid-base pair. To test the hypothesis that cysteine 230 and lysine 238 act as an acid-base pair to deprotonate biotin, site-directed mutagenesis was used to mutate cysteine 230 to alanine (C230A) and lysine 238 to glutamine (K238Q). Mutations at either residue resulted in a 50-fold increase in the K(m) for ATP. The C230A mutation had no effect on the formation of carboxybiotin, indicating that cysteine 230 does not play a role in the deprotonation of biotin. However, the K238Q mutation resulted in no formation of carboxybiotin, which showed that lysine 238 has a role in the carboxylation reaction. N-Ethylmaleimide was found to inactivate the C230A mutant but not the K238Q mutant, suggesting that N-ethylmaleimide is reacting with lysine 238 and not cysteine 230. The pH dependence of N-ethylmaleimide inactivation revealed that the pK value for lysine 238 was 9.4 or higher, suggesting lysine 238 is not a catalytic base. Thus, the results suggest that cysteine 230 and lysine 238 do not act as an acid-base pair in the deprotonation of biotin.
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PMID:Do cysteine 230 and lysine 238 of biotin carboxylase play a role in the activation of biotin? 1074 3

Biotin carboxyl carrier protein (BCCP) is the small biotinylated subunit of Escherichia coli acetyl-CoA carboxylase (ACC), the enzyme that catalyzes the first committed step of fatty acid synthesis. Similar proteins are found in other bacteria and in chloroplasts. E. coli BCCP is a member of a large family of protein domains modified by covalent attachment of biotin to a specific lysine residue. However, the BCCP biotinyl domain differs from many of these proteins in that an eight-amino acid residue insertion is present upstream of the biotinylated lysine. X-ray crystallographic and multidimensional NMR studies show that these residues constitute a structure that has the appearance of an extended thumb that protrudes from the otherwise highly symmetrical domain structure. I report that expression of two mutant BCCPs lacking the thumb residues fails to restore growth and fatty acid synthesis to a temperature-sensitive E. coli strain that lacks BCCP when grown at nonpermissive temperature. Alignment of BCCPs from various organisms shows that only two of the eight thumb residues are strictly conserved, and amino acid substitution of either residue results in proteins giving only weak growth of the temperature-sensitive E. coli strain. Therefore, the thumb structure is essential for the function of BCCP in the ACC reaction and provides a useful motif for distinguishing the biotinylated proteins of multisubunit ACCs from those of enzymes catalyzing other biotin-dependent reactions. An unexpected result was that expression of a mutant BCCP in which the biotinylated lysine residue was substituted with cysteine was able to partially restore growth and fatty acid synthesis to the temperature-sensitive E. coli strain. This complementation was shown to be specific to BCCPs having native structure (excepting the biotinylated lysine) and is interpreted in terms of dimerization of the BCCP biotinyl domain during the ACC reaction.
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PMID:The biotinyl domain of Escherichia coli acetyl-CoA carboxylase. Evidence that the "thumb" structure id essential and that the domain functions as a dimer. 1149 22

Fatty acid synthesis in pea chloroplasts is regulated by light/dark. The regulatory enzyme acetyl-CoA carboxylase is modulated by light/dark, presumably under redox regulation. Acetyl-CoA carboxylase is a multienzyme complex composed of biotin carboxylase and carboxyltransferase (CT). To demonstrate the redox regulation of CT, composed of the nuclear-encoded alpha and the chloroplast-encoded beta subunits, we identified the cysteine residues involved in such regulation. We expressed the recombinant CT in Escherichia coli and found that the partly deleted CT was, like the full-length CT, sensitive to a redox state. Site-directed mutagenesis of the deleted CT showed that replacement by alanine of the cysteine residue 267 in the alpha polypeptide or 442 in the beta polypeptide resulted in redox-insensitive CT and broke the intermolecular disulfide bond between the alpha and beta polypeptides. Similar results were confirmed in the full-length CT. These results indicate that the two cysteines in recombinant CT are involved in redox regulation by intermolecular disulfide-dithiol exchange between the alpha and beta subunits. Immunoblots of extract from plants incubated in the light or dark supported that such a disulfide-dithiol exchange is relevant in vivo. A covalent bond between a nuclear-encoded polypeptide and a chloroplast-encoded polypeptide probably regulates the enzyme activity in response to light.
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PMID:Thiol-disulfide exchange between nuclear-encoded and chloroplast-encoded subunits of pea acetyl-CoA carboxylase. 1154 65

Glucose transport is stimulated in a variety of cells and tissues in response to inhibition of oxidative phosphorylation. However, the underlying mechanisms and mediating steps remain largely unknown. In the present study we first tested whether a decrease in the redox state of the cell per se and the resultant increase in generation of reactive oxygen species (ROS) lead to stimulation of glucose transport. Clone 9 cells (expressing the Glut1 isoform of facilitative glucose transporters) were exposed to azide, lactate, and ethanol for 1 h. Although all three agents stimulated glucose transport and increased cell NADH-to-NAD(+) ratio and phospho-ERK1/2, signifying increased ROS generation, the response to the stimuli was not blocked by N-acetyl-l-cysteine (an agent that counteracts ROS); moreover, the response to azide was not blocked by diamide (an intracellular sulfhydryl oxidizing agent). We then found that cell AMP-to-ATP and ADP-to-ATP ratios were increased and 5'-AMP-activated protein kinase (AMPK) was stimulated by all three agents, as evidenced by increased phosphorylation of AMPK and acetyl-CoA carboxylase. We conclude that although azide, lactate, and ethanol increase NADH-to-NAD(+) ratios and ROS production, their stimulatory effect on glucose transport is not mediated by increased ROS generation. However, all three agents increased cell AMP-to-ATP ratio and stimulated AMPK, making it likely that the latter pathway plays an important role in the glucose transport response.
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PMID:Role of 5'-AMP-activated protein kinase in stimulation of glucose transport in response to inhibition of oxidative phosphorylation. 1616 57

The pyruvate dehydrogenase complex from pea (Pisum sativum L.) mitochondria was purified 23-fold by high speed centrifugation and glycerol gradient fractionation. The complex had a s(20,w) of 47.5S but this is a minimal value since the complex is unstable. The complex is specific for NAD(+) and pyruvate; NADP(+) and other keto acids give no reaction. Mg(2+), thiamine pyrophosphate, and cysteine are also required for maximal activity. The pH optimum for the complex was between 6.5 and 7.5.Continuous sucrose density gradients were used to separate castor bean (Ricinus communis L.) endosperm proplastids from mitochondria. Pyruvate dehydrogenase complex activity was found to be coincident with the proplastid peak on all of the gradients. Some separation of proplastids and mitochondria could be achieved by differential centrifugation and the ratios of the activities of the pyruvate dehydrogenase complex to succinic dehydrogenase and acetyl-CoA carboxylase to succinic dehydrogenase were consistent with both the pyruvate dehydrogenase complex and acetyl-CoA carboxylase being present in the proplastid. The proplastid fraction has to be treated with a detergent, Triton X-100, before maximal activity of the pyruvate dehydrogenase complex activity is expressed, indicating that it is bound in the organelle. The complex had a sharp pH optimum of 7.5. The complex required added Mg(2+), cysteine, and thiamine pyrophosphate for maximal activity but thiamine pyrophosphate was inhibitory at higher concentrations.
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PMID:Pyruvate dehydrogenase complex from higher plant mitochondria and proplastids. 1665 53

There is evidence that reactive oxygen species (ROS) signalling is required for normal increases in glucose uptake during contraction of isolated mouse skeletal muscle, and that AMP-activated protein kinase (AMPK) is involved. The aim of this study was to determine whether ROS signalling is involved in the regulation of glucose disposal and AMPK activation during moderate-intensity exercise in humans. Nine healthy males completed 80 min of cycle ergometry at 62 +/- 1% of peak oxygen consumption ( V(O(2)peak).A 6,6-(2)H-glucose tracer was infused at rest and during exercise, and in a double-blind randomised cross-over design, N-acetylcysteine (NAC) or saline (CON) was co-infused. NAC was infused at 125 mg kg(1) h(1) for 15 min and then at 25 mg kg(1) h(1) for 20 min before and throughout exercise. NAC infusion elevated plasma NAC and cysteine, and muscle NAC and cysteine concentrations during exercise. Although neither NAC infusion nor exercise significantly affected muscle reduced or oxidised glutathione (GSH or GSSG) concentration (P > 0.05), S-glutathionylation (an indicator of oxidative stress) of a protein band of approximately 270 kDa was increased approximately 3-fold with contraction and this increase was prevented by NAC infusion. Despite this, exercised-induced increases in tracer determined glucose disposal, plasma lactate, plasma non-esterified fatty acids (NEFAs), and decreases in plasma insulin were not affected by NAC infusion. In addition, skeletal muscle AMPKalpha and acetyl-CoA carboxylase-beta (ACCbeta) phosphorylation increased during exercise by approximately 3- and approximately 6-fold (P < 0.05), respectively, and this was not affected by NAC infusion. Unlike findings in mouse muscle ex vivo, NAC does not attenuate skeletal muscle glucose disposal or AMPK activation during moderate-intensity exercise in humans.
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PMID:N-Acetylcysteine infusion does not affect glucose disposal during prolonged moderate-intensity exercise in humans. 2030 50


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