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

---

Query: EC:6.2.1.1 (ACS)
78,556 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bioluminescence provides a sensitive analytical approach for the measurement of low concentrations of ATP and can be used to monitor any reaction in which ATP is consumed or synthesized. We describe a quick, sensitive method involving bioluminescence for the quantitation of chloramphenicol in serum. Chloramphenicol acetyl transferase catalyzes the acetylation of chloramphenicol in the presence of acetyl coenzyme A. ATP is consumed when the acetylation reaction is coupled to the acetyl coenzyme A synthetase reaction. Residual ATP is then assayed with the firefly luciferin-luciferase reaction. The procedure can be completed in less than 1 h and requires as little as 20 microliter of serum. The response of the assay is linear with concentration through a range of 2 to 20 mg/L and shows good correlation with a gas-chromatographic method (r = 0.978) and a radioenzymic method (r = 0.985). No significant interference was found from five other antibiotics tested. The small sample requirement makes the assay especially applicable to infant and pediatric monitoring, where the effects of toxicity are greatest.
...
PMID:A bioluminescence micromethod for measuring chloramphenicol in serum. 723 58

The mutant gene coding for a proline-activating domain (grs2-pro) was cloned and sequenced from Bacillus brevis Nagano, BII-3 strain, which produces gramicidin S synthetase 2 defective in proline-activation. By comparison of the nucleotide sequence with the wild-type sequence, a single point mutation was found at the 2609th guanine, which was replaced with adenine, resulting in the change of the 870th glycine to glutamic acid. Homology search for the deduced amino acid sequence of grs2-pro gene revealed that the 870th glycine was conserved in adenylate-forming enzymes, and its flanking sequence was highly conserved among the aminoacyl adenylate-forming enzymes, such as antibiotic peptide synthetases: gramicidin S synthetase 1 and 2 (GS1, GS2), tyrocidine synthetase 1 (TS1), and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS); and other aminoacyl adenylation enzymes: alpha-aminoadipate reductase (LYS2), EntF, and AngR. On the other hand, this flanking sequence was not conserved in the other adenylate-forming enzymes lacking amino acid activation, such as acetyl-CoA synthetase, long-chain acyl-CoA synthetase, luciferase, and 4-coumarate CoA ligase. Single base substitutions at the 870th GGG codon were carried out by oligonucleotide site-directed mutagenesis. Four mutagenized clones were isolated, containing grs2-pro genes which exchange 870-Gly for alanine, valine, arginine, and tryptophan. The translated products from these clones could scarcely catalyze proline-dependent ATP-32PPi exchange reaction. The coil structure of 870-Gly region was lost in the mutants. These results suggest that the 870-Gly residue of grs2-pro protein is essential for aminoacyl-adenylation in the antibiotic peptide synthetase family.
...
PMID:Effect of single base substitutions at glycine-870 codon of gramicidin S synthetase 2 gene on proline activation. 827 62

This paper describes a spectrophotometric assay that can measure the inorganic pyrophosphate produced from various enzymatic reactions. This is a coupled assay in which the addition of inorganic pyrophosphatase initially cleaves the pyrophosphate into two molecules of phosphate. The phosphate is then detected by the conversion of 2-amino-6-mercapto-7-methylpurine ribonucleoside to 2-amino-6-mercapto-7-methylpurine by purine nucleoside phosphorylase [M.R. Webb (1992) Proc. Natl. Acad. Sci. USA 89, 4884-4887]. The reaction is monitored by measuring the increase in absorbance at 360 nm. The generation of two molecules of phosphate from each molecule of pyrophosphate increases the sensitivity of the assay, which has a linear range from about 1 to 75 nmol pyrophosphate in a 1-ml reaction volume. To demonstrate the general usefulness of this assay, we show that the inorganic pyrophosphate generated by reactions involving acetyl-CoA synthetase and luciferase can be readily detected and that continuous as well as end-point assays can be performed.
...
PMID:A spectrophotometric method to measure enzymatic activity in reactions that generate inorganic pyrophosphate. 895 23

The superfamily of adenylate forming enzymes including peptide synthetases, acyl-CoA synthetases and insect luciferases is readily identified by the signature sequence SGTTGXPKG. This sequence including an invariant lysyl residue is located in a disordered loop region and was predicted to be of significant antigenicity. Antibodies were generated employing YTSGTTGRPKGC attached to bovine serum albumin and have been successfully used to identify respective enzymes and adenylate forming domains in multienzyme systems. These include the delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetases of Aspergillus nidulans and Acremonium chrysogenum, gramicidin S synthetase 1 and tyrocidine synthetase 1 from Bacillus brevis, acetyl-CoA synthetase from Alcaligenes eutrophus and a putative peptide synthetase from Metarhizium anisopliae. Weaker or no reactions are observed when the amino acid in position X in the protein is non-basic or hydrophobic, which is respectively the case for gramicidin S synthetase 1 and luciferase.
...
PMID:Group specific antibodies against the putative AMP-binding domain signature SGTTGXPKG in peptide synthetases and related enzymes. 953 May 7

1. Formation of acyl-coenzymes (Co)A occurs as an obligatory step in the metabolism of a variety of endogenous substrates, including fatty acids. The reaction is catalysed by ATP-dependent acid:CoA ligases (EC 6.2.1.1-2.1.3; AMP forming), classified on the basis of their ability to conjugate saturated fatty acids of differing chain lengths, short (C2-C4), medium (C4-C12) and long (C10-C22). The enzymes are located in various cell compartments (cytosol, smooth endoplasmic reticulum, mitochondria and peroxisomes) and exhibit wide tissue distribution, with highest activity associated with liver and adipose tissue. 2. Formation of acyl-CoA is not unique to endogenous substrates, but also occurs as an obligatory step in the metabolism of some xenobiotic carboxylic acids. The mitochondrial medium-chain CoA ligase is principally associated with metabolism via amino acid conjugation and activates substrates such as benzoic and salicylic acids. Although amino acid conjugation was previously considered an a priori route of metabolism for xenobiotic-CoA, it is now recognized that these highly reactive and potentially toxic intermediates function as alternative substrates in pathways of intermediary metabolism, particularly those associated with lipid biosyntheses. 3. In addition to a role in fatty acid metabolism, the hepatic microsomal and peroxisomal long-chain-CoA-ligases have been implicated in the formation of the acyl-CoA thioesters of a variety of hypolipidaemic and peroxisome proliferating agents (e.g. clofibric acid) and of the R(-)-enantiomers of the commonly used 2-arylpropionic acid non-steroidal anti-inflammatory drugs (e.g. ibuprofen). In vitro kinetic studies using rat hepatic microsomes and peroxisomes have alluded to the possibility of xenobiotic-CoA ligase multiplicity. Although cDNA encoding a long-chain ligase have been isolated from rat and human liver, there is currently no molecular evidence of multiple isoforms. The gene has been localized to chromosome 4 and homology searches have revealed a significant similarity with enzymes of the luciferase family. 4. Increasing recognition that formation of a CoA conjugate increases chemical reactivity of xenobiotic carboxylic acids has led to an awareness that the relative activity, substrate specificity and intracellular location of the xenobiotic-CoA ligases may explain differences in toxicity. 5. Continued characterization of the human xenobiotic-CoA ligases in terms of substrate/inhibitor profiles and regulation, will allow a greater understanding of the role of these enzymes in the metabolism of carboxylic acids.
...
PMID:Role of hepatic fatty acid:coenzyme A ligases in the metabolism of xenobiotic carboxylic acids. 978 15

The findings presented here originally arose from the suggestion that the synthesis of dinucleoside polyphosphates (Np(n)N) may be a general process involving enzyme ligases catalyzing the transfer of a nucleotidyl moiety via nucleotidyl-containing intermediates, with release of pyrophosphate. Within this context, the characteristics of the following enzymes are presented. Firefly luciferase (EC 1.12. 13.7), an oxidoreductase with characteristics of a ligase, synthesizes a variety of (di)nucleoside polyphosphates with four or more inner phosphates. The discrepancy between the kinetics of light production and that of Np(n)N synthesis led to the finding that E*L-AMP (L = dehydroluciferin), formed from the E*LH(2)-AMP complex (LH(2) = luciferin) shortly after the onset of the reaction, was the main intermediate in the synthesis of (di)nucleoside polyphosphates. Acetyl-CoA synthetase (EC 6.2.1.1) and acyl-CoA synthetase (EC 6.2.1. 8) are ligases that synthesize p(4)A from ATP and P(3) and, to a lesser extent, Np(n)N. T4 DNA ligase (EC 6.5.1.1) and T4 RNA ligase (EC 6.5.1.3) catalyze the synthesis of Np(n)N through the formation of an E-AMP complex with liberation of pyrophosphate. DNA is an inhibitor of the synthesis of Np(n)N and conversely, P(3) or nucleoside triphosphates inhibit the ligation of a single-strand break in duplex DNA catalyzed by T4 DNA ligase, which could have therapeutic implications. The synthesis of Np(n)N catalyzed by T4 RNA ligase is inhibited by nucleoside 3'(2'),5'-bisphosphates. Reverse transcriptase (EC 2.7.7.49), although not a ligase, catalyzes, as reported by others, the synthesis of Np(n)ddN in the process of removing a chain termination residue at the 3'-OH end of a growing DNA chain.
...
PMID:Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase and several ligases. 1100 93

T4 DNA ligase and the ubiquitin activating enzyme (E1), catalyze the synthesis of ATP beta,gamma-bisphosphonate derivatives. Concerning T4 DNA ligase: (i) etidronate (pC(OH)(CH(3))p) displaced the AMP moiety of the complex E-AMP in a concentration dependent manner; (ii) the K(m) values and the rate of synthesis k(cat) (s(-1)), determined for the following compounds were, respectively: etidronate, 0.73+/-0.09 mM and (70+/-10)x10(-3) s(-1); clodronate (pCCl(2)p), 0.08+/-0.01 mM and (4.1+/-0.3)x10(-3) s(-1); methylenebisphosphonate (pCH(2)p), 0.024+/-0.001 mM and (0.6+/-0.1)x10(-3) s(-1); tripolyphosphate (P(3)) (in the synthesis of adenosine 5'-tetraphosphate, p(4)A), 1.30+/-0.30 mM and (6.2+/-1.1)x10(-3) s(-1); (iii) in the presence of GTP and ATP, inhibition of the synthesis of Ap(4)G was observed with clodronate but not with pamidronate (pC(OH)(CH(2)-CH(2)-NH(3))p). Concerning the ubiquitin activating enzyme (E1): methylenebisphosphonate was the only bisphosphonate, out of the ones tested, that served as substrate for the synthesis of an ATP derivative (K(m)=0.36+/-0.09 mM and k(cat)=0.15+/-0.02 s(-1)). None of the above bisphosphonates were substrates of the reaction catalyzed by luciferase or by acyl-CoA synthetase. The ability of acetyl-CoA synthetase to use methylenebisphosphonate as substrate depended on the commercial source of the enzyme. In our view this report widens our knowledge of the enzymes able to metabolize bisphosphonates, a therapeutic tool widely used in the treatment of osteoporosis.
...
PMID:Synthesis of bisphosphonate derivatives of ATP by T4 DNA ligase, ubiquitin activating enzyme (E1) and other ligases. 1837 15

Bioactive small molecules, including steroids, activate multiple signaling pathways in mammalian cells. However, current technologies cannot illuminate such multiple effects of a ligand in mammalian cells. Here, we demonstrate integrated-molecule-format multicolor systems simultaneously visualizing bifacial activities of a ligand, where estrogen receptor alpha (ERalpha) was exemplified to demonstrate the present technology. First, we developed a single-molecule-format probe emitting red bioluminescence for imaging interaction between the phosphorylated ligand binding domain of ERalpha (ER LBD) and the Src homology-2 (SH2) domain of Src. The SH2 domain-linked ER LBD was sandwiched between dissected N- and C-terminal fragments of Pyrophorus plagiophthalamus (click beetle) luciferase emitting red bioluminescence. Second, another single-molecule-format bio-luminescent probe emitting green bioluminescence was constructed to visualize intramolecular interaction between ER LBD and LXXLL motifs. Mammalian cells carrying the two probes emit red and/or green light in response to agonistic and antagonistic activities of a ligand, which correspond to its genomic and nongenomic activities, respectively. Third, the two probes were assembled to make an single-molecule-format multicolor indicator, in which all of the components for ligand sensing and multiple-light emission were integrated. The probe emitted characteristic light spectra in response to various agonists and antagonists. This is the first example where (i) protein phosphorylation was recognized with a single bioluminescent probe and (ii) bifacial activities of a ligand, either agonistic or antagonistic, were simultaneously visualized with multiple colors.
ACS Chem Biol 2008 Jun 20
PMID:An integrated-molecule-format multicolor probe for monitoring multiple activities of a bioactive small molecule. 1857 Mar 55

The importance of bioluminescence in enabling a broad range of high-throughput screening (HTS) assay formats is evidenced by widespread use in industry and academia. Therefore, understanding the mechanisms by which reporter enzyme activity can be modulated by small molecules is critical to the interpretation of HTS data. In this Perspective, we provide evidence for stabilization of luciferase by inhibitors in cell-based luciferase reporter-gene assays resulting in the counterintuitive phenomenon of signal activation. These data were derived from our analysis of luciferase inhibitor compound structures and their prevalence in the Molecular Libraries Small Molecule Repository using 100 HTS experiments available in PubChem. Accordingly, we found an enrichment of luciferase inhibitors in luciferase reporter-gene activation assays but not in assays using other reporters. In addition, for several luciferase inhibitor chemotypes, we measured reporter stabilization and signal activation in cells that paralleled the inhibition determined using purified luciferase to provide further experimental support for these contrasting effects.
ACS Chem Biol 2008 Aug 15
PMID:A specific mechanism for nonspecific activation in reporter-gene assays. 1870 52

Signaling via the androgen receptor (AR) plays an important role in human health and disease. All currently available anti-androgens prevent ligand access to the receptor, either by limiting androgen synthesis or by competitive antagonism at the ligand binding domain. It is unknown to what extent various steps of receptor activation may be separable and distinctly targeted by inhibitors. We have previously described the use of fluorescent protein fusions to AR to monitor its subcellular distribution and ligand-induced conformational change by fluorescence resonance energy transfer (FRET). We have now used a microscopy-based screen to identify inhibitors that prevent AR conformational change or nuclear accumulation after ligand activation. Hits were secondarily selected on the basis of their ability to inhibit AR transcription at a PSA-luciferase promoter and were tested for effects on (3)H-DHT binding to AR in cells. We find a strong correlation between compounds that block DHT binding and those that inhibit nuclear accumulation. These compounds are structurally distinct from known antagonists. Additional compounds blocked AR conformational change but did not affect DHT binding or nuclear localization of AR. One compound increased ligand-induced FRET yet functioned as a potent inhibitor. These results suggest that multiple inhibitory conformations of AR are possible and can be induced by diverse mechanisms. The lead compounds described here may be candidates for the development of novel antiandrogens and may help identify new therapeutic targets.
ACS Chem Biol 2009 Mar 20
PMID:AR inhibitors identified by high-throughput microscopy detection of conformational change and subcellular localization. 1923 99


1 2 3 4 5 6 7 8 9 10 Next >>

---