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)

The biotin carboxyl carrier protein (BCCP) component of Escherichia coli acetyl coenzyme A carboxylase and three peptides derived from BCCP by proteolytic digestion have been examined by circular dichroism spectroscopy. BCCP, which has a peptide molecular weight of 22,500, has a spectrum typical of globular proteins with negative extrema at 222 nm and 208 nm. The two smallest peptides, BCCP(SC) and BCCP(9,100), with molecular weights of 8,900 and 9,100, respectively, exhibit unusual positive CD bands centered at 237 nm and 220 nm. BCCP(10,400), with a molecular weight of 10,400, has a CD spectrum intermediate between BCCP and that of the smallest peptides. Since d-biotin exhibits a positive CD band at 233 nm, it was suspected that the biotin prosthetic group might be the chromophore responsible for the 237 nm CD band seen in BCCP(SC) and BCCP(9,100). Enzymatic carboxylation of BCCP(SC) to form CO2-BCCP(SC) caused the CD spectrum to change with a shift of the 237 nm band to 232 nm. The positive CD band at 220 nm was unaffected by carboxylation of the biotin prosthetic group. These date suggest that the 237 nm signal may be due either to the biotin which acts as a chromophore directly or to a chromophore that is perturbed by the carboxylation of biotin. A spectropolarimetric titration was carried out to investigate the possible contribution of the single tyrosine residue of BCCP(SC) to the CD spectrum of this peptide. At pH values over 9 the CD spetrum changed with the disappearance of the 237 nm band, suggesting that tyrosine might contribute to this CD band. Denaturation of BCCP(SC) or BCCP(9,100) with 8 M urea of 6 M guanidine HCl abolished the positive CD bands and resulted in spectra typical of a random coil, whereas treatment of BCCP(SC) with 1% sodium dodecyl sulfate abolished the positive bands and left a spectrum exhibiting a shoulder at 222 nm and a negative band at 205 nm, suggestive of a high degree of ordered structure. It is concluded that the CD band at 237 nm in BCCP(SC) and BCCP(9,100) is prabably due to a noncovalent interaction of biotin with an amino acid residue(s) of the protein. It is suggested that the biotin prosthetic group is partially buried in the surface of the protein, rather than swinging free at the end of the lysine side chain through which it is covalently linked to the protein, to permit this interaction to occur.
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PMID:Acetyl coenzyme A carbosylase. Circular dichroism studies of Escherichia coli biotin carboxyl carrier protein. 0 38

The interaction of rat liver acetyl-CoA carboxylase with a 2',3'-dialdehyde derivative of ATP (oATP) has been studied. The degree of the enzyme inactivation has been found to depend on the oATP concentration and the incubation time. ATP was proved to be the only substrate which protected the inactivation. Acetyl-CoA did not effect inactivation, while HCO3- accelerated the process. Ki values for oATP in the absence and presence of HCO3- were 0.35 +/- 0.04 and 0.5 +/- 0.06 mM, and those of the modification constant (kmod) were 0.11 and 0.26 min-1 respectively. oATP completely inhibited the [14C]ADP in equilibrium ATP exchange and did not effect the [14C]acetyl-CoA in equilibrium malonyl-CoA exchange. Incorporation of approximately 1 equivalent of [3H]oATP per acetyl-CoA carboxylase subunit has been shown. No recovery of the modified enzyme activity has been observed in Tris or beta-mercaptoethanol containing buffers, and treatment with NaB3H4 has not led to 3H incorporation. The modification elimination of the ATP triphosphate chain. The results indicated the affinity modification of acetyl-CoA carboxylase by oATP. It was shown that the reagent apparently interacted selectively with the epsilon-amino group of lysine in the ATP-binding site to form a morpholine-like structure.
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PMID:Affinity labelling of rat liver acetyl-CoA carboxylase by a 2',3'-dialdehyde derivative of ATP. 196 47

The biotin-binding site of acetyl-CoA carboxylase from rat was characterized as to its amino acid sequence and relative position in the enzyme molecule. Biotin binds to the lysyl residue in the tetrapeptide Val-Met-Lys-Met; this tetrapeptide is located in close proximity to the NH2 terminus. In all other biotin-containing enzymes, the conserved tetrapeptide Ala-Met-Lys-Met is the counterpart to that of rat acetyl-CoA carboxylase; and the lysyl residue is 35 residues from the COOH terminus. To examine the significance of these unusual features of the biotinylation site of animal acetyl-CoA carboxylase, cDNA fragments were expressed in a bacterial system and the effects of specific site-directed mutagenesis were examined. Replacement of Val by Ala in the conserved tetrapeptide abolished biotinylation of the expressed protein. However, introduction of a termination codon at residue 36, in such a way that the distance between the lysine on which biotin binds and the COOH-terminal amino acid was 35 residues and the penultimate amino acid was the hydrophobic residue leucine, increased the efficiency of biotinylation, provided a substantial portion of the NH2-terminal peptide was removed.
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PMID:Analysis of the biotin-binding site on acetyl-CoA carboxylase from rat. 256 68

The interaction of rat liver acetyl-CoA carboxylase with a 2',3'-dialdehyde derivative of ATP (oATP) has been studied. The degree of the enzyme inactivation has been found to depend on the oATP concentration and the incubation time. ATP was the only reaction substrate which provided protection from inactivation. Acetyl-CoA did not affect inactivation, while HCO3- accelerated the process. Ki values for oATP in the absence and the presence of HCO3- were 0.35 +/- 0.04 and 0.5 +/- 0.06 mM, and those of the modification constant (k) were 0.11 and 0.26 min-1, respectively. oATP completely inhibited the reaction of [14C]ADP in equilibrium ATP exchange, whereas produced actually no effect on [14C]acetyl-CoA equilibrium with malonyl-CoA exchange. Incorporation of about one equivalent of [3H]oATP per acetyl-CoA carboxylase subunit has been shown. No restoration of the modified enzyme activity has been observed in Tris or beta-mercaptoethanol containing buffers, and treatment with NaB[3H]4 has not led to 3H incorporation. The modification process involves elimination of the triphosphate chain of oATP. The results obtained indicate the affinity character of oATP-mediated modification of acetyl-CoA carboxylase. The reagent apparently interacts selectively with the epsilon-amino group of lysine in the ATP-binding site to form a morpholine-like structure.
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PMID:[Acetyl-CoA-carboxylase: modification of ATP-binding site of the active center by 2',3'-dialdehyde derivative of ATP]. 257 82

Biotin-dependent carboxylases require covalently bound biotin for enzymatic activity. The biotin is attached through a lysine residue, which in a number of bacterial, avian, and mammalian carboxylases, is found within the conserved sequence Ala-Met-Lys-Met. We have determined the partial nucleotide sequence of cDNA clones for human propionyl-CoA carboxylase and pyruvate carboxylase. The predicted amino acid sequence of both these proteins contains the conserved tetrapeptide 35 residues from the carboxy terminus. In addition, both proteins contain the tripeptide, Pro-Met-Pro, 26 residues toward the amino terminus from the biotin attachment site. The overall amino acid homology through this region is 43%. Similar findings have been made for the biotin-containing polypeptides of transcarboxylase of Propionibacterium shermanii and acetyl-CoA carboxylase of Escherichia coli (W. L. Maloy, B. U. Bowien, G. K. Zwolinski, K. G. Kumar, and H. G. Wood (1979) J. Biol. Chem. 254, 11615-11622). The implications of this sequence conservation with regard to the function and evolution of biotin-dependent carboxylases is discussed. We propose that the 60 amino acids surrounding the biotin site are bounded by a proline "hinge" and the carboxy terminus has remained conserved as a result of constraints imposed by biotinylation of the enzyme.
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PMID:Sequence homology around the biotin-binding site of human propionyl-CoA carboxylase and pyruvate carboxylase. 355 48

Definitive evidence is presented for the bifunctional nature of the biotin repressor protein which possesses both regulatory and enzymatic activities. The repressor protein can activate biotin in the presence of ATP to form biotinyl-5'-adenylate, the co-repressor which remains tightly bound to the repressor protein. This complex can either bind to the operator site and inhibit transcription or transfer the biotinyl moiety to a lysine residue of the apoenzyme of acetyl-CoA carboxylase. The two activities were coincident throughout a purification procedure which resulted in a 3500-fold increase in activity. Gel electrophoresis of the purified preparation, under native or denaturing conditions, showed three proteins with the activity corresponding to the major protein band of apparent Mr = 34,000. On gel exclusion chromatography, the activity was also associated with a protein of Mr varying fro 37,000-44,000, indicating the protein is monomeric. The occasional appearance of multiple bands with biological activity in the native gels suggests that the repressor protein can also exist in multimeric forms. On chromatofocusing, the repressor activity and the holoenzyme synthetase activity were coincidental, with the peak of activity at pH 7.2, the isoelectric point. Only a single protein band with Mr = 34,000 was observed on SDS gel electrophoresis of all fractions showing activity.
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PMID:Purification and properties of the biotin repressor. A bifunctional protein. 612 46

Acetyl-coenzyme A carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-coenzyme A, which is utilized in the plastid for de novo fatty acid synthesis and outside the plastid for a variety of reactions, including the synthesis of very long chain fatty acids and flavonoids. Recent evidence for both multifunctional and multisubunit ACCase isozymes in dicot plants has been obtained. We describe here the isolation of a tobacco (Nicotiana tabacum L. cv bright yellow 2 [NT1]) cDNA clone (E3) that encodes a 58.4-kD protein that shares 80% sequence similarity and 65% identity with the Anabaena biotin carboxylase subunit of ACCase. Similar to other biotin carboxylase subunits of acetyl-CoA carboxylase, the E3-encoded protein contains a putative ATP-binding motif but lacks a biotin-binding site (methionine-lysine-methionine or methionine-lysine-leucine). The deduced protein sequence contains a putative transit peptide whose function was confirmed by its ability to direct in vitro chloroplast uptake. The subcellular localization of this biotin carboxylase has also been confirmed to be plastidial by western blot analysis of pea (Pisum sativum), alfalfa (Medicago sativa L.), and castor (Ricinus communis L.) plastid preparations. Northern blot analysis indicates that the plastid biotin carboxylase transcripts are expressed at severalfold higher levels in castor seeds than in leaves.
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PMID:Structural analysis, plastid localization, and expression of the biotin carboxylase subunit of acetyl-coenzyme A carboxylase from tobacco. 761 Jan 68

The complete amino acid sequence of 3T3-L1 adipocyte pyruvate carboxylase (PC) [pyruvate:carbon-dioxide ligase (ADP-forming), EC 6.4.1.1] has been deduced from sequencing overlapping cDNA clones obtained from an adipocyte cDNA library constructed in the lambda Zap vector. The encoding mRNA for PC promoter contains 4067 nt, including a 3534-nt coding sequence and noncoding regions of 100 and 433 nt at the 5' and 3' ends, respectively. The biotinylated lysine of the encoded PC promoter (1178 amino acids with a calculated M(r) of apocarboxylase = 129,784) is located 35 residues from the COOH-terminal end and, as in most other biotin enzymes, is in the consensus sequence AMKM. The adipocyte PC is closely similar (53% identity) to the yeast enzyme and contains different segments that are homologous with regions from the biotin carboxylase component of Escherichia coli acetyl-CoA carboxylase, the keto acid-binding subunits of Propionibacterium shermanii oxaloacetate transcarboxylase and Klebsiella pneumoniae oxaloacetate decarboxylase, and to the biotin carboxyl-carrier protein of the bacterial biotin enzymes. In addition to the putative mitochondrial targeting signal, functional domains are readily identifiable in the sequence and are in the following order: biotin carboxylase-carboxyltransferase-biotin carboxyl-carrier protein, as proposed for yeast PC.
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PMID:Adipose pyruvate carboxylase: amino acid sequence and domain structure deduced from cDNA sequencing. 844 88

The Escherichia coli repressor of biotin biosynthesis (BirA) is a unique transcriptional repressor which catalyzes synthesis of its own corepressor and catalyzes attachment of a cofactor to an essential metabolic enzyme. BirA both catalyzes synthesis of biotinyl-5'-AMP from the substrates ATP and biotin and transfer of the biotin moiety from the adenylate to a lysine residue of a subunit of the acetyl-CoA carboxylase. BirA-bio-5'-AMP, moreover, binds sequence specifically to the biotin operator to repress transcription of the biotin biosynthetic genes. Using a combination of kinetic measurements of binding of the two ligands, biotin and bio-5'-AMP, to BirA as well as proteolytic digestion experiments, we have found evidence for at least three discrete conformational states of BirA. Results of stopped-flow fluorescence measurements of association of both ligands with BirA indicate that the process involves initial formation of a collision complex followed by a slow conformational change. The kinetics of the conformational change are distinct for the two ligands and are the basis for the difference in the thermodynamic stabilities of the two protein-ligand complexes. Different rates of proteolytic digestion of apoBirA and complexes of BirA with the two ligands were also observed. Results of the combined approaches indicate that apoBirA, and the BirA-bio-5'-AMP and BirA-biotin complexes are conformationally distinct.
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PMID:Evidence for distinct ligand-bound conformational states of the multifunctional Escherichia coli repressor of biotin biosynthesis. 852 35

Biotin biosynthesis and retention in Escherichia coli is regulated by the multifunctional protein, BirA. The protein acts as both the transcriptional repressor of the biotin biosynthetic operon and as a ligase for covalent attachment of biotin to a unique lysine residue of the acetyl-CoA carboxylase. Biotinyl-5'-AMP is the activated intermediate for the ligase reaction and the allosteric effector for DNA binding. We have purified and characterized apoBCCP and a truncated form containing the COOH-terminal 87 residues (apoBCCP87). Molecular masses of the proteins measured using matrix-assisted laser desorption ionization time-of-flight mass spectrometry conformed to the expected values. The assembly states of apoBCCP and apoBCCP87 were determined using sedimentation equilibrium ultracentrifugation. Nearly quantitative enzymatic transfer of biotin from BirA-biotinyl-5'-AMP to the apoBCCP forms was assessed using two methods, mass spectrometric analysis of acceptor proteins after incubation with BirA-bio-5'-AMP and a steady state fluorescence assay. The BirA catalyzed rates of transfer of biotin from bio-5'-AMP to apoBCCP and apoBCCP87 were measured by stopped-flow fluorescence. Kinetic parameters estimated from these measurements indicate that the intact and truncated forms of the acceptor protein are functionally identical.
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PMID:Purification and characterization of intact and truncated forms of the Escherichia coli biotin carboxyl carrier subunit of acetyl-CoA carboxylase. 863 88


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