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1.
O Froyshov 《Acta microbiologica Academiae Scientiarum Hungaricae》1975,22(4):427-432
The dodecapeptide bacitracin A is the major constitutent of a family of antibacterial peptides produced by Bacillus licheniformis. The non-ribosomal biosynthesis of bacitracin has been studied in cell-free extracts. Bacitracin synthetase has been fractionated on Sephadex G 200 column into two fractions; both fractions were required for bacitracin biosynthesis. On the other hand, on a Sepharose affinity chromatography column, using L-leucine as ligand, three fractions were obtained; all three were required for bacitracin biosynthesis. During bacitracin synthesis, the enzyme components contain a number of thioester bound peptides. The nature of the peptides suggested that the synthesis proceeds towards the C-terminal end of the molecule. It is assumed that by sequential addition of thioester-bound amino acids, bacitracin A could be synthesized on the surface of the enzyme containing phosphopantetheine. 相似文献
2.
The map position of a mutation in the bacitracin synthetase gene(s) in Bacillus licheniformis ATCC 10716 was determined by transduction with phage SP-15. Results indicate that it is linked to the lys and trp loci and is distinct from the known sporulation loci on the chromosome of Bacillus licheniformis. The defect(s) of the enzyme complex were analysed in terms of its ability to bind covalently 14C-labelled amino acid precursors of the bacitracin molecule. 相似文献
3.
Peptides at the tRNA binding site of the crystallizable monomeric form of E. coli methionyl-tRNA synthetase.
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A protein affinity labeling derivative of E. coli tRNA(fMet) carrying lysine-reactive cross-linking groups has been covalently coupled to monomeric trypsin-modified E. coli methionyl-tRNA synthetase. The cross-linked tRNA-synthetase complex has been isolated by gel filtration, digested with trypsin, and the tRNA-bound peptides separated from the bulk of the free tryptic peptides by anion exchange chromatography. The bound peptides were released from the tRNA by cleavage of the disulfide bond of the cross-linker and purified by reverse-phase high-pressure liquid chromatography, yielding three major peptides. These peptides were found to cochromatograph with three peptides of known sequence previously cross-linked to native methionyl-tRNA synthetase through lysine residues 402, 439 and 465. These results show that identical lysine residues are in close proximity to tRNA(fMet) bound to native dimeric methionyl-tRNA synthetase and to the crystallizable monomeric form of the enzyme, and indicate that cross-linking to the dimeric protein occurs on the occupied subunit of the 1:1 tRNA-synthetase complex. 相似文献
4.
Mycobacillin synthetase lacks aspartic acid racemase, alanine racemase and glutamic acid racemase activities. The enzyme also does not respond to ATP-[32P]Pi exchange, nor does it catalyse the antibiotic synthesis in presence of amino acids of configuration opposite to that present in the molecule. Preincubation with optical isomers of opposite configuration inhibited the ATP-[32P]Pi exchange reaction to the extent of 60-90%. None of the three fractions of mycobacillin synthetase contained a pantothenic acid arm. Two molecules of ATP are required to synthesize one peptide bond of mycobacillin. Intermediate peptides of mycobacillin are not covalently linked to the three-fraction mycobacillin synthetase. 相似文献
5.
Formylglycinamide ribonucleotide synthetase from Escherichia coli: cloning, sequencing, overproduction, isolation, and characterization 总被引:11,自引:0,他引:11
The purL gene of Escherichia coli encoding the enzyme formylglycinamidine ribonucleotide (FGAM) synthetase which catalyzes the conversion of formylglycinamide ribonucleotide (FGAR), glutamine, and MgATP to FGAM, glutamate, ADP, and Pi has been cloned and sequenced. The mature protein, as deduced by the structural gene sequence, contains 1628 amino acids and has a calculated Mr of 141,418. Comparison of the purL control region to other pur loci control regions reveals a common region of dyad symmetry which may be the binding site for the "putative" repressor protein. Construction of an overproducing strain permitted purification of the protein to homogeneity. N-Terminal sequence analysis and comparison of glutamine binding domain sequences (Ebbole & Zalkin, 1987) confirm the amino acid sequence deduced from the gene sequence. The purified protein exhibits glutaminase activity of 0.02% the normal turnover, and NH3 can replace glutamine as a nitrogen donor with a Km = 1 M and a turnover of 3 min-1 (2% glutamine turnover). The enzyme forms an isolable (1:1) complex with glutamine: t1/2 is 22 min at 4 degrees C. This isolated complex is not chemically competent to complete turnover when FGAR and ATP are added, demonstrating that ammonia and glutamine are not covalently bound as a thiohemiaminal available to complete the chemical conversion to FGAM. hydroxylamine trapping experiments indicate that glutamine is bound covalently to the enzyme as a thiol ester. Initial velocity and dead-end inhibition kinetic studies on FGAM synthetase are most consistent with a sequential mechanism in which glutamine binds followed by rapid equilibrium binding of MgATP and then FGAR. Incubation of [18O]FGAR with enzyme, ATP, and glutamine results in quantitative transfer of the 18O to Pi. 相似文献
6.
Sarvagya S. Katiyar David Pan John W. Porter 《Biochemical and biophysical research communications》1982,104(2):517-522
S-(4-bromo-2,3-dioxobutyl)-CoA has been used as an inhibitor of fatty acid synthetase from pigeon liver. This affinity label selectively and irreversibly inhibits the acetyl transacylase and β-ketoacyl synthetase reactions of this multienzyme complex. Binding studies with [3H]-labeled bromodioxobutyl-CoA have established that four mol of the inhibitor are bound per mol of the enzyme complex, and that the radioactivity of this compound is covalently bound to cysteine and 4′-phosphopantetheine moieties. Other partial reactions of fatty acid synthesis are unaffected by bromodioxobutyl-CoA. 相似文献
7.
A Gadow J Vater W Schlumbohm Z Palacz J Salnikow H Kleinkauf 《European journal of biochemistry》1983,132(2):229-234
The reactive thioester complexes of gramicidin S synthetase with substrate amino acids and intermediate peptides are slowly hydrolyzed in neutral buffer solutions under mild conditions. Fully active enzyme is recovered. These processes are strongly accelerated by certain thiol protective agents. In the presence of 1 mM dithioerythritol the half-life times of these hydrolysis reactions are in the range of 1-90 h at 3 degrees C. The thioester complex of gramicidin S synthetase 2 (GS2, the heavy enzyme) with the tripeptide DPhe-Pro-Val is distinguished by the highest stability of all these intermediates. A different decomposition pattern is observed for the thioester complex of GS2 with LOrn. Here 3-amino-2-piperidone (cyclo-LOrn) is formed in a rapid cyclization reaction. This product specifically blocks the activation center of GS2 for LOrn at the thioester binding site. All other activation reactions of gramicidin S synthetase are unaffected. A procedure for a specific labelling of the reaction centers of the multienzyme is outlined. 相似文献
8.
Effect of decoyinine on peptidoglycan synthesis and turnover in Bacillus subtilis. 总被引:7,自引:3,他引:4
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The sporulation of Bacillus subtilis can be induced in the presence of amino acids and glucose by partially depriving the cells of guanine nucleotides. This can be achieved, e.g., by the addition of decoyinine, a specific inhibitor of GMP synthetase. To determine the effect of this and other inhibitors on cell wall synthesis, we measured in their presence the incorporation of acetylglucosamine into acid-precipitable material. The rate of wall synthesis decreased by 50% within 5 min after decoyinine addition; this decrease was prevented by the presence of guanosine. A comparison with the effects of other inhibitors of cell wall synthesis indicated that decoyinine inhibited the final portion of the cell wall biosynthetic pathway, i.e., after the steps inhibited by bacitracin or vancomycin. Decoyinine addition also prevented cellular autolysis and cell wall turnover. It is not known whether these two effects of decoyinine on cell wall synthesis are causally related. 相似文献
9.
10.
A method for the preparation of an activated protein carrier is described: Protein carboxyl groups are transformed into N-hydroxysulfosuccinimide esters, a structure that will react with primary amino groups under amide bond formation. Although the activated ester is unstable under aqueous conditions, a significant amount of hapten molecules can be bound covalently to the carrier under very mild conditions. Ligands can be peptides or other molecules possessing a primary amino group. The method avoids the risk of ligand polymerization and no derivatization of the ligand prior to conjugation is needed. Residual unreacted ligand molecules can therefore be recovered in their native form by size exclusion chromatography. The method was used to conjugate two synthetic sugar beet acetolactate synthetase (E.C. 4.1.3.18) peptides to diphtheria toxoid. Antibodies were raised against both of the conjugates. The specificity of these antibodies against sugar beet acetolactate synthetase was verified using immunoblotting, ELISA, and immunoprecipitation. 相似文献
11.
Raney nickel (Ni(H)) catalyzes a specific reductive cleavage of carbon-sulfur bonds and, therefore, can be used to determine whether compounds are covalently bound to proteins through a sulfide linkage. When the covalent thymidylate synthetase-[3H]5-fluoro-2'-deoxyuridylic acid-[14C]-5,10-CH2H4-folate complex (Langenbach et al. (1972a), Biochem, Biophys. Res. Commun. 48, 1565) was denatured and then shaken with Ni(H) at 25 degrees C, both isotopes were rapidly cleaved from the protein, with identical reaction halftimes of less than 10 min. The liberated radioactivity was filterable through nitro-cellulose filters and comigrated with small molecules on Sephadex G-25. Both labels migrated identically upon paper chromatography. A [3H]5-fluoro-2'-deoxyuridylic acid-[35S]thymidylate synthetase complex was formed with enzyme isolated from Lactobacillus casei grown in the presence of [35S]cysteine. This complex, upon Ni(H) treatment, released both tritium and sulfur-35 at identical rates. Control experiments on amino acids showed that only the sulfur-containing amino acids are degraded by Ni(H). Cysteine was rapidly converted to alanine and methionine to alpha-aminobutyric acid. 5-Carboxymethylcysteine and 5-uracilylcysteine, simple models for the tenary enzyme-5-fluoro-2'-deoxyuridylic acid-5,10-CH2H4-folate complex, were converted to alanine at the same rate that 5-fluoro-2'-deoxyuridylic acid (FdUrd-5'-P) was cleaved from the enzyme. Native ribonuclease, which has a tightly coiled structure, was not affected by the reagent, but carboxymethylated ribonuclease was desulfurized. Amino acid analysis of Ni(H)-treated thymidylate synthetase showed that cysteine was the only amino acid degraded. Gel electrophoresis of the proteins after exposure to Ni(H) showed no breakage of polypeptide chains. These results support a sulfide linkage between FdUrd-5'-P and thymidylate synthetase in the covalent complex. 相似文献
12.
Cotranslational attachment of fatty acids to nascent peptides in gastric mucus glycoprotein 总被引:1,自引:0,他引:1
B L Slomiany H Tsukada A Slomiany 《Biochemical and biophysical research communications》1986,141(1):387-393
Using gastric mucous cells which are involved exclusively in the synthesis of secretory O-glycosidic glycoprotein (mucin), the relationship between protein core synthesis and its acylation with fatty acids was investigated. Labeling of the cells with [3H]palmitic acid and [35S]methionine followed by isolation of peptidyl-tRNA and release of nascent peptides, indicated that these peptides contain covalently bound fatty acids. The high performance thin layer chromatography, SDS-gel electrophoresis, and radioactivity scanning revealed that the preparation contained three fractions labeled with palmitate (Mr 15,000-3,600) and two (Mr 1,500 and less) without this label. Based on these data and the nascent peptides amino acid analysis, we conclude that the protein core of the O-glycosidic glycoprotein is acylated with fatty acids during translation, when the peptide chain is longer than 21 amino acid residues. 相似文献
13.
The aminoacyl-tRNA synthetases covalently link transfer RNAs to their cognate amino acids. Some of the tRNA synthetases have employed an editing mechanism to ensure fidelity in this first step of protein synthesis. The amino acid editing active site for Escherichia coli leucyl-tRNA synthetase resides within the CP1 domain that folds discretely from the main body of the enzyme. A portion of the editing active site is lined with conserved threonines. Previously, we identified one of these threonine residues (Thr(252)) as a critical amino acid specificity factor. On the basis of X-ray crystal structure information, two other nearby threonine residues (Thr(247) and Thr(248)) were hypothesized to interact with the editing substrate near its cleavage site. Single mutations of either of these conserved threonine residues had minimal effects on amino acid editing. However, double mutations that deleted the hydroxyl group from the neighboring threonine residues abolished amino acid editing activity. We propose that these threonine residues, which are also conserved in the homologous isoleucyl-tRNA synthetase and valyl-tRNA synthetase editing active sites, play a central role in amino acid editing. It is possible that they collaborate in stabilizing the transition state. 相似文献
14.
15.
Actinomycin synthetases. Multifunctional enzymes responsible for the synthesis of the peptide chains of actinomycin 总被引:6,自引:0,他引:6
U Keller 《The Journal of biological chemistry》1987,262(12):5852-5856
Two enzymes were purified from actinomycin-synthesizing Streptomyces chrysomallus which could be identified as peptide synthetases involved in the biosynthesis of actinomycin. Actinomycin synthetase II activates the first two amino acids of the peptide chains of the peptide lactone antibiotic, threonine and valine (or isoleucine), as thioesters via their corresponding adenylates. It is a single polypeptide chain of Mr 225,000. Similarly, actinomycin synthetase III activates proline, glycine, and valine (the remaining three amino acids in the antibiotic) as thioesters and is a single polypeptide chain of about Mr 280,000. It also carries the methyltransferase function(s) for N-methylation of thioesterified glycine and valine. In addition, it catalyzes the formation of cyclo(sarcosyl-N-methyl-L-valine) from glycine, L-valine, and S-adenosyl-L-methionine at the expense of ATP. Although the cell-free synthesis of the peptide lactone was not as yet accomplished, the data provide evidence that together with the 4-methyl-3-hydroxyanthranilic acid-activating enzyme (now designated as actinomycin synthetase I) all amino acid-activating protein components of the actinomycin-synthesizing enzyme complex are identified. 相似文献
16.
The influence of certain L-amino acids and their mixtures on the synthesis of exoprotease from Bacillus thuringiensis was studied. Physiological experiments showed that the mixture of 20 amino acids added to the artificial medium repressed the synthesis of exoprotease. Among the compounds studied there are both the compounds which stimulate the synthesis of exoprotease (glutamic and aspartic acids, glycine), and the compounds which repress the synthesis of the enzyme (proline, tryptophane, tyrosine, asparagine, serine, cystein). None of the amino acids caused a change in the exoprotease activity. It has been assumed that the repression of the protease synthesis in the presence of the amino acids is accomplished by ammonium ions, which are formed when using the amino acids of Bac. thuringiensis. The glutamine synthetase activity of cells was determined during the growth of Bac. thuringiensis both on a medium containing triptone and after the addition of certain amino acids to the cell suspension. The correlation between the influence of different amino acids on the synthesis of exoprotease and the glutamine synthetase activity was demonstrated. 相似文献
17.
Regulation of Synthesis of the Aminoacyl-Transfer Ribonucleic Acid Synthetases for the Branched-Chain Amino Acids of Escherichia coli 总被引:10,自引:10,他引:0
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The regulation of synthesis of valyl-, leucyl-, and isoleucyl-transfer ribonucleic acid (tRNA) synthetases was examined in strains of Escherichia coli and Salmonella typhimurium. When valine and isoleucine were limiting growth, the rate of formation of valyl-tRNA synthetase was derepressed about sixfold; addition of these amino acids caused repression of synthesis of this enzyme. The rate of synthesis of the isoleucyl- and leucyl-tRNA synthetases was derepressed only during growth restriction by the cognate amino acid. Restoration of the respective amino acid to these derepressed cultures caused repression of synthesis of the aminoacyl-tRNA synthetase, despite the resumption of the wild-type growth rate. 相似文献
18.
19.
Purification and properties of serine hydroxymethyltransferase from Sulfolobus solfataricus.
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Serine hydroxymethyltransferase (SHMT) catalyzes the reversible cleavage of serine to glycine with the transfer of the one-carbon group to tetrahydrofolate to form 5,10-methylenetetrahydrofolate. No SHMT has been purified from a nonmethanogenic Archaea strain, in part because this group of organisms uses modified folates as the one-carbon acceptor. These modified folates are not readily available for use in assays for SHMT activity. This report describes the purification and characterization of SHMT from the thermophilic organism Sulfolobus solfataricus. The exchange of the alpha-proton of glycine with solvent protons in the absence of the modified folate was used as the activity assay. The purified protein catalyzes the synthesis of serine from glycine and a synthetic derivative of a fragment of the natural modified folate found in S. solfataricus. Replacement of the modified folate with tetrahydrofolate did not support serine synthesis. In addition, this SHMT also catalyzed the cleavage of both allo-threonine and beta-phenylserine in the absence of the modified folate. The cleavage of these two amino acids in the absence of tetrahydrofolate is a property of other characterized SHMTs. The enzyme contains covalently bound pyridoxal phosphate. Sequences of three peptides showed significant similarity with those of peptides of SHMTs from two methanogens. 相似文献
20.
A new bifunctional affinity label, 5'-p-(fluorosulfonyl)benzoyl-8-azidoadenosine (5'-FSBAzA), has been synthesized by condensation of p-(fluorosulfonyl)benzoyl chloride with 8-azidoadenosine. 5'-FSBAzA has been characterized by elemental analysis, thin-layer chromatography, and ultraviolet and 1H NMR spectroscopy. The affinity label contains both an electrophilic fluorosulfonyl moiety and a photoactivatable azido group which are capable of reacting with several classes of amino acids found in enzymes. 5'-FSBAzA reacts with bovine liver glutamate dehydrogenase in a two-step process: a dark reaction yielding about 0.5 mol of the sulfonylbenzoyl-8-azidoadenosine (SBAzA) group bound/mol enzyme subunit by reaction of the enzyme at the fluorosulfonyl group, followed by photolysis in which 25% of the covalently bound SBAzA becomes crosslinked to the enzyme. 5'-FSBAzA-modified glutamate dehydrogenase, both before and after photolysis, retains full catalytic activity but is less sensitive to allosteric inhibition by GTP, to activation by ADP, and to inhibition by 1 mM NADH. These results suggest the modification in the dark reaction of a regulatory nucleotide binding site. Photoactivation of the covalently bound reagent may have general applicability in relating modified amino acids which are close to each other in the region of the purine nucleotide binding sites of glutamate dehydrogenase and other proteins. 相似文献