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1.
A highly conserved protein motif characteristic of Class II aminoacyl tRNA synthetases was found to align with a region of Escherichia coli asparagine synthetase A. The alignment was most striking for aspartyl tRNA synthetase, an enzyme with catalytic similarities to asparagine synthetase. To test whether this sequence reflects a conserved function, site-directed mutagenesis was used to replace the codon for Arg298 of asparagine synthetase A, which aligns with an invariant arginine in the Class II aminoacyl tRNA synthetases. The resulting genes were expressed in E. coli, and the gene products were assayed for asparagine synthetase activity in vitro. Every substitution of Arg298, even to a lysine, resulted in a loss of asparagine synthetase activity. Directed random mutagenesis was then used to create a variety of codon changes which resulted in amino acid substitutions within the conserved motif surrounding Arg298. Of the 15 mutant enzymes with amino acid substitutions yielding soluble enzyme, 13 with changes within the conserved region were found to have lost activity. These results are consistent with the possibility that asparagine synthetase A, one of the two unrelated asparagine synthetases in E. coli, evolved from an ancestral aminoacyl tRNA synthetase. 相似文献
2.
Dinescu A Cundari TR Bhansali VS Luo JL Anderson ME 《The Journal of biological chemistry》2004,279(21):22412-22421
Glutathione synthetase is an enzyme that belongs to the glutathione synthetase ATP-binding domain-like superfamily. It catalyzes the second step in the biosynthesis of glutathione from gamma-glutamylcysteine and glycine in an ATP-dependent manner. Glutathione synthetase has been purified and sequenced from a variety of biological sources; still, its exact mechanism is not fully understood. A variety of structural alignment methods were applied and four highly conserved residues of human glutathione synthetase (Glu-144, Asn-146, Lys-305, and Lys-364) were identified in the binding site. The function of these was studied by experimental and computational site-directed mutagenesis. The three-dimensional coordinates for several human glutathione synthetase mutant enzymes were obtained using molecular mechanics and molecular dynamics simulation techniques, starting from the reported crystal structure of human glutathione synthetase. Consistent with circular dichroism spectroscopy, our results showed no major changes to overall enzyme structure upon residue mutation. However, semiempirical calculations revealed that ligand binding is affected by these mutations. The key interactions between conserved residues and ligands were detected and found to be essential for enzymatic activity. Particularly, the negatively charged Glu-144 residue plays a major role in catalysis. 相似文献
3.
The effect of nordihydroguaiaretic acid and related lignans on formyltetrahydrofolate synthetase and carboxylesterase 总被引:1,自引:0,他引:1
The lignans nordihydroguaiaretic acid (NDGA), heminordihydroguaiaretic acid (HNDGA) and norisoguaiacin were found to inhibit formyltetrahydrofolate synthetase (formate:tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) and carboxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1) activity from a wide variety of sources. In all cases, NDGA was the most effective inhibitor. Synthetase activity was reduced by half at NDGA concentrations between 0.11 and 0.24 mM. Esterase activity consisted of NDGA-sensitive and NDGA-resistant forms. The sensitive class was half-inhibited by 2-4 microM NDGA. Irreversible inhibition of formyltetrahydrofolate synthetase by NDGA was observed both at low protein concentration (less than 0.2 mg/ml) and at high protein concentration where precipitation of protein was observed. Inhibition of formyltetrahydrofolate synthetase by NDGA arises from a decrease in Vmax and increase in Km for all substrates. In contrast, NDGA affects only the Vmax parameter of the esterase activity. It is suggested that the broad range of enzymes inhibited by NDGA may be a consequence of the amphipathic character of the molecule and the flexibility to accommodate to a variety of binding sites. It is also suggested that the previously reported ability of NDGA to inhibit phagocytosis may be due to the compound's ability to inhibit carboxylesterases. 相似文献
4.
2-5A synthetase is an important component of the mammalian antiviral 2-5A system. At present, the existence of 2-5A synthetase in the lowest animals, the marine sponges, has been demonstrated, although this enzyme has not been found in bacteria, yeast or plants. Here, we studied the 2-5A synthesizing capacity and the product profile of a variety of marine sponges belonging to Demospongia subclasses Tetractinomorpha and Ceractinomorpha. The 2-5A synthetase activity varied largely, in the range of four orders of magnitude, depending on the sponge species. Compared with the enzymes of the mammalian 2-5A synthetase family, the most active sponge species exhibited a surprisingly high 2-5A synthetase specific activity. Unlike the mammalian 2-5A synthetases that produce 2-5A oligomers in the presence of a double-stranded RNA activator, the 2-5A synthetase(s) from sponges were active without the addition of dsRNA. The sponge species differed in their product profiles. A novel product pool formed by Chondrosia reniformis was identified as a series of long 2-5A oligomers (up to 17-mers) with the prevalence of heptamers and octamers. The large variability of qualitative and quantitative characteristics of sponge 2-5A synthetases may refer to the occurrence of a variety of 2-5A synthetase isozymes in sponges. 相似文献
5.
Selection and properties of Escherichia coli mutants defective in the synthesis of cyclopropane fatty acids.
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Mutants of Escherichia coli K-12 defective in the synthesis of cyclopropane fatty acids (CFA) have been selected and isolated by a L-[methyl-3H]methionine suicide procedure. Two mutants were isolated. Stationary-phase cultures of both mutants contain less than 0.7% of the CFA content found in the parental strain. The CFA deficiency is attributed to a deficiency of CFA synthetase activity. Extracts of both mutants contain less than 10% of the CFA synthetase activity found in extracts of the parental strain. Experiments in which parental and mutant extracts were mixed indicate that the lack of activity in the mutant strains is not due to an inhibitor of CFA synthetase present in the mutant extracts. We have not yet detected a physiological phenotype for these mutants. These strains grow normally at various temperatures in a variety of media. We have tested survival (colony-forming ability) in response to (i) prolonged incubation in stationary phase, (ii) exposure to drying, and (iii) exposure to detergents, heavy metals, low pH, high salt concentration, and a variety of other environmental conditions. The survival of both mutants is identical to that of the parental strain under all conditions tested. The compositions (excepting the CFA deficiency) and metabolic turnover rates of the phospholipids of both mutant strains are indistinguishable from those of the wild-type strain. The transport of several amino acids also seems normal in these mutants. 相似文献
6.
Mutant strains of Salmonella typhimurium with defective phosphoribosylpyrophosphate synthetase activity 总被引:4,自引:0,他引:4
A mutant of Salmonella typhimurium with undetectable phosphoribosylpyrophosphate (PRPP) synthetase activity in vitro and abnormally low PRPP pools in vivo was identified by screening temperature-sensitive isolates by an autoradiographic procedure. The lack of PRPP synthetase activity in vitro and temperature-sensitive growth were shown to result from separate, but closely linked mutations mapping at 47 units on the Salmonella chromosome. Mutant cell extracts prepared by a variety of methods did not show any detectable PRPP synthetase activity, but material that was immunochemically cross-reactive with PRPP synthetase was detected by complement fixation analysis. A second mutant, isolated by localized mutagenesis, contained about half the PRPP synthetase and cross-reacting material of the parental strain. 相似文献
7.
The properties of the histamine metabolizing enzyme, gamma-glutamylhistamine synthetase (gamma-GHA synthetase) were studied in Aplysia ganglia in vitro. This enzyme catalyzes the incorporation of histamine into peptide linkage with L-glutamate to form a peptidoamine, gamma-glutamylhistamine (gamma-GHA). gamma-GHA synthetase is a soluble enzyme with an apparent Km of 653 microM for histamine and 10.6 mM for L-glutamate. Synthesis of gamma-GHA is energy-dependent, having an absolute requirement for ATP. Magnesium ions and dithiothreitol are also essential for activity. Of a variety of gamma-glutamyl compounds and glutamate analogs tested, only L-glutamate was effectively incorporated into peptide linkage with histamine. Similarly, the enzyme has a higher affinity for histamine than for numerous imidazole analogs. In addition, 3,4-dihydroxyphenylethylamine (dopamine), 5-hydroxytryptamine, octopamine, and several other amines tested are effective inhibitors of gamma-GHA synthesis. Ganglia, nerve trunks, and the capsule surrounding the ganglion had the highest synthetase activity. The specific activity of the enzyme in muscle, heart, and hemolymph was less than 10% of that in ganglia. Differences in substrate specificity and effect of inhibitors distinguish gamma-GHA synthetase from gamma-glutamyl transpeptidase, glutamine synthetase, and carnosine synthetase. 相似文献
8.
Cartilage cyclic nucleotide phosphodiesterase: inhibition by anti-inflammatory agents 总被引:8,自引:0,他引:8
Soluble 3′,5′-nucleotide phosphodiesterase (PDE) activity is described in chicken epiphyseal and articular cartilage. Kinetic studies of these enzymes demonstrate a high and low Km for the substrates, adenosine 3′,5′-cyclic monophosphate (cyclic AMP) and guanosine 3′,5′-cyclic monophosphate (cyclic GMP). Epiphyseal and articular PDE activities are inhibited by those anti-inflammatory agents which are potent inhibitors of the enzyme, prostaglandin synthetase (PS). Specificity of this inhibition is indicated by the activity of these agents against the low Km enzyme. Other anti-inflammatory agents with significantly less potency as PS inhibitors or with no activity against prostaglandin synthetase are found to be either inactive or relatively less potent as inhibitors of cartilage PDE activity. A variety of other anti-inflammatory or anti-rheumatic agents, which are not known to affect prostaglandin synthetase activity, are poor inhibitors of cartilage PDE activity. These data provide insight into the mechanism of action of certain anti-inflammatory agents and into the relationships between prostaglandins and inflammatory reactions. 相似文献
9.
Role of glutamine synthetase activity in the uptake and metabolism of arginine and proline in the cyanobacterium Anabaena cycadeae 总被引:1,自引:0,他引:1
Surendra Singh 《FEMS microbiology letters》1993,106(3):335-340
Abstract The uptake of arginine and proline and their assimilation as nitrogen source have been studied in the cyanobacterium Anabaena cycadeae and its glutamine auxotropic mutant lacking glutamine synthetase activity. The uptake pattern of arginine and proline was found to be biphasic in both wild-type and mutant strains, consisting of an initial fast phase lasting up to 60 s followed by a slower second phase. The uptake activities of both the amino acids were also found to be similar in both the strains. The wild-type strain, having normal glutamine synthetase activity, utilized arginine and proline as sole nitrogen source, whereas the mutant strain lacking glutamine synthetase activity could not do so. These results suggest that: (1) glutamine synthetase activity is necessarily required for the assimilation of arginine and proline as nitrogen source, but it is not required for the uptake of these amino acids; and (2) glutamine synthetase serves as the sole ammonia-assimilating enzyme as well as glutamine-forming route in heterocystous cyanobacteria. 相似文献
10.
Homogenates of seminiferous tubules from rat testes catalyzed the incorporation of label from [14C]isopentenyl diphosphate into a variety of polyprenyl products. Long chain polyprenyl mono- and diphosphates were formed as major products when undesirable side reactions were minimized. The long chain polyprenyl diphosphate synthetase was measured as a sum of the mono- and diphosphate derivatives formed and was dependent on the addition of t,t-farnesyl diphosphate, isopentenyl diphosphate, and divalent cation. The highest activity was associated with the membranous fractions, whereas activity was negligible in the cytosolic fraction. The products of this prenyl transferase were labile to acid and yielded petroleum ether soluble products which indicated that the alpha-isoprene unit was unsaturated. Hydrolysis of either the polyprenyl mono-or diphosphates with a testicular phosphatase in the absence of NaF yielded C75, C80, C85, and C90 polyprenols. The chain lengths of the products of the synthetase suggest that this enzyme is responsible for the de novo biosynthesis of dehydrodolichyl diphosphates which are precursors of the dolichyl derivatives found in testes. 相似文献
11.
Of a variety of purine and pyrimidine nucleotides tested, only ADP and 5'AMP significantly inhibited the Mg(2+)-dependent activity of pea leaf glutamine synthetase. They were less effective inhibitors where Mn(2+) replaced Mg(2+). They were competitive inhibitors with respect to ATP, with inhibition constant (Ki) values of 1.2 and 1.8 mm, respectively. The energy charge significantly affects the activity of glutamine synthetase, especially with Mg(2+). Of a variety of amino acids tested, l-histidine and l-ornithine were the most inhibitory, but significant inhibition was seen only where Mn(2+) was present. Both amino acids appeared to compete with l-glutamate, and the Ki values were 1.9 mm for l-histidine (pH 6.2) and 7.8 mm for l-ornithine (pH 6.2). l-Alanine, glycine, and l-serine caused slight inhibition (Mn(2+)-dependent activity) and were not competitive with ATP or l-glutamate.Carbamyl phosphate was an effective inhibitor only when Mn(2+) was present, and did not compete with substrates. Inorganic phosphate and pyrophosphate caused significant inhibition of the Mg(2+)-dependent activity. 相似文献
12.
The location of nitrite reductase and other enzymes related to amino Acid biosynthesis in the plastids of root and leaves 总被引:25,自引:14,他引:11
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Miflin BJ 《Plant physiology》1974,54(4):550-555
Density gradient separation of plastids from leaf and root tissue was carried out. The distribution in the gradients of the activity of the following enzymes was determined: nitrite reductase, glutamine synthetase, acetolactate synthetase, aspartate aminotransferase, catalase, cytochrome oxidase, and triosephosphate isomerase. The distribution of chlorophyll was followed in gradients from leaf tissue. The presence of plastids that have retained their stroma enzymes was denoted by a peak of triosephosphate isomerase activity. Coincidental with this peak were bands of nitrite reductase, acetolactate synthetase, glutamine synthetase, and aspartate aminotransferase activity. The results suggest that most, if not all, the nitrite reductase and acetolactate synthetase activity of the cell is in the plastids. The plastids were found to contain only part of the total glutamine synthetase, aspartate aminotransferase, and triosephosphate dehydrogenase activity in the cell. Some evidence was obtained for low levels of glutamate dehydrogenase activity in chloroplasts. 相似文献
13.
Hitoshi Kusakabe Kenjiro Kodama Haruhiko Machida Akira Kuninaka 《Bioscience, biotechnology, and biochemistry》2013,77(9):1983-1984
A variety of bacteria and yeasts were examined for activities of biotin biosynthetic enzymes, including pimelyl-CoA synthetase, 7-keto-8-aminopelargonic acid (KAPA) synthetase, 7,8-diaminopelargonic acid (DAPA) aminotransferase and dethiobiotin (DTB) synthetase. Among the strains tested, only Bacillus sphaericus, a DTB producer, showed significant activities for all four enzymes. The bacterium also exhibited high activity of biotin synthesis from DTB in an intact cell system. Using cell-free extract and intact cells, some properties of DAPA aminotransferase, DTB synthetase and biotin synthesizing reaction were examined.Based on these results of enzyme activities DTB productivity of B. sphaericus was discussed. 相似文献
14.
In order to provide some insight into the mechanism of hyaluronate synthesis, the subcellular localization of the synthetase system for hyaluronate was determined in eukaryotic cells. The mouse oligodendroglioma cell line G26-24, which produces copious amounts of hyaluronate in culture, was chosen as a system for these studies. Protease treatment and homogenization of cells followed by hyaluronate synthetase assay suggested that nucleotide-binding sites and trypsin-sensitive synthetase sites were not exposed at the outer membrane surface. Protease treatment following homogenization did result in decreased activity. Membrane fragments, prepared by gentle homogenization in iso- and hypotonic buffers, were subjected to differential centrifugation followed by several continuous and discontinuous sucrose equilibrium and velocity gradient systems. Hyaluronate synthetase activity co-fractionated with a plasma membrane marker in all systems, including those in which Golgi markers were separable. Treatment of intact cells in culture with several hyaluronidases resulted in a marked stimulation of cell-free synthetase activity. The stimulated activity was also found exclusively in plasma membrane-enriched fractions. 相似文献
15.
Kinetic studies of the fatty acid synthetase multienzyme complex from Euglena gracilis variety bacillaris.
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A fatty acid synthetase multienzyme complex was purified from Euglena gracilis variety bacillaris. The fatty acid synthetase activity is specifically inhibited by antibodies against Escherichia coli acyl-carrier protein. The Euglena enzyme system requires both NADPH and NADH for maximal activity. An analysis was done of the steady-state kinetics of the reaction catalysed by the fatty acid synthetase multienzyme complex. Initial-velocity studies were done in which the concentrations of the following pairs of substrates were varied: malonyl-CoA and acetyl-CoA, NADPH and acetyl-CoA, malonyl-CoA and NADPH. In all three cases patterns of the Ping Pong type were obtained. Product-inhibition studies were done with NADP+ and CoA. NADP+ is a competitive inhibitor with respect to NADPH, and uncompetitive with respect to malonyl-CoA and acetyl-CoA. CoA is uncompetitive with respect to NADPH and competitive with respect to malonyl-CoA and acetyl-CoA. When the concentrations of acetyl-CoA and malonyl-CoA were varied over a wide range, mutual competitive substrate inhibition was observed. When the fatty acid synthetase was incubated with radiolabelled acetyl-CoA or malonyl-CoA, labelled acyl-enzyme was isolated. The results are consistent with the idea that fatty acid synthesis proceeds by a multisite substituted-enzyme mechanism involving Ping Pong reactions at the following enzyme sites: acetyl transacylase, malonyl transacylase, beta-oxo acyl-enzyme synthetase and fatty acyl transacylase. 相似文献
16.
Effects of univalent cations on the activity of particulate starch synthetase 总被引:2,自引:2,他引:0
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An investigation was made to determine the univalent cation requirements of starch synthetase from a variety of plant species of economic importance. The particulate enzyme from sweet corn was shown to have an absolute requirement for potassium, with the optimum activation occurring at 0.05 M KCl. Rubidium, cesium, and ammonium were 80% as effective as potassium while sodium and lithium were respectively 21% and 8% as effective as potassium. The KA for potassium was determined to be 6 mM. In the case of the particulate starch synthetase from wheat, bush beans, field corn, soybeans, peas, or potatoes, considerable stimulation of enzyme activity was obtained by the addition of potassium to the reaction mixture. In these studies, low enzyme activity was observed in the absence of added potassium, but the content of endogenous univalent cations in the reactions may be sufficient to account for the activities observed. Anions of various types had no effect on starch synthetase activity. Divalent cations produced slight activation in the presence or absence of potassium. All efforts to show a potassium requirement for glycogen synthetase from rat liver have been negative. 相似文献
17.
Regulation of urease and ammonia assimilatory enzymes in Selenomonas ruminantium. 总被引:4,自引:3,他引:1
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Urease and glutamine synthetase activities in Selenomonas ruminantium strain D were highest in cells grown in ammonia-limited, linear-growth cultures or when certain compounds other than ammonia served as the nitrogen source and limited the growth rate in batch cultures. Glutamate dehydrogenase activity was highest during glucose (energy)-limited growth or when ammonia was not growth limiting. A positive correlation (R = 0.96) between glutamine synthetase and urease activities was observed for a variety of growth conditions, and both enzyme activities were simultaneously repressed when excess ammonia was added to ammonia-limited, linear-growth cultures. The glutamate analog methionine sulfoximine (MSX), inhibited glutamine synthetase activity in vitro, but glutamate dehydrogenase, glutamate synthase, and urease activities were not affected. The addition of MSX (0.1 to 100 mM) to cultures growing with 20 mM ammonia resulted in growth rate inhibition that was dependent upon the concentration of MSX and was overcome by glutamine addition. Urease activity in MSX-inhibited cultures was increased significantly, suggesting that ammonia was not the direct repressor of urease activity. In ammonia-limited, linear-growth cultures, MSX addition resulted in growth inhibition, a decrease in GS activity, and an increase in urease activity. These results are discussed with respect to the importance of glutamine synthetase and glutamate dehydrogenase for ammonia assimilation under different growth conditions and the relationship of these enzymes to urease. 相似文献
18.
Hemolysate uroporphyrinogen (uro) I synthetase activity was found to be inhibited by lead chloride; whereas enzymatic activity in hepatic cytosol was unaffected. Dialysis of hepatic cytosol or purification of the enzyme rendered uro I synthetase activity sensitive to lead. Inhibition of uro I synthetase activity by lead was non-competitive and reversible for both hemolysate and hepatic cytosol. Using gel chromatography, a factor was separated from uro I synthetase activity in both hemolysate and hepatic cytosol that protected against lead inhibition of enzymatic activity. A higher concentration of factor was found in hepatic cytosol than in hemolysate, which provides an explanation for the differential inhibition of uro I synthetase activity by lead for these two tissues. This factor is heat stable, but is destroyed by acid, base, ashing or by preincubation with protease. No free sulfhydryl content was detected in purified factor preparations. Metallothionein, isolated from rat liver, was incapable of protecting against lead inhibition of uro I synthetase activity. These findings indicate that the factor is probably protein in nature, but that it is distinctly different from metallothionein. 相似文献
19.
The activity of uridine diphosphate glucose-d-fructose 6-phosphate 2-glucosyltransferase in leaves
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Hawker JS 《The Biochemical journal》1967,105(3):943-946
1. By using EDTA in reaction mixtures it was possible to determine the activity of sucrose phosphate synthetase in freshly prepared leaf extracts without the complications caused by sucrose phosphatase. 2. EDTA was found also to increase the activity of sucrose phosphate synthetase by as much as 100%. 3. High sucrose phosphate synthetase activities were found in leaf preparations in which sucrose phosphatase was inhibited by EDTA. By contrast with previous reports, the activities were sufficient to allow sucrose synthesis in leaves during photosynthesis to occur via sucrose phosphate. 4. Sugar-cane plants having different rates of photosynthesis also had different activities of sucrose phosphate synthetase in their leaves. 5. It is suggested that the activity of sucrose phosphate synthetase in leaves may play a role in the control of the rate of photosynthesis. 相似文献
20.
Chinese hamster ovary cells resistant to borrelidin overproduce threonyl-tRNA synthetase 总被引:2,自引:0,他引:2
Chinese hamster ovary cell lines that are 1000-fold more resistant to the threonyl-tRNA synthetase inhibitor borrelidin than the sensitive parental cells were isolated after stepwise selection for growth in increasing concentrations of the drug. These cells show a 10-20-fold increase in threonyl-tRNA synthetase activity. Quantitation of the amount of threonyl-tRNA synthetase protein by immunological techniques indicated a 60-100-fold increase compared to sensitive cells. No significant changes in the Km for substrates, inhibition by borrelidin or thermal stability were found for the threonyl-tRNA synthetase of resistant cells. These data suggest that the resistant cell lines may have amplified the gene encoding threonyl-tRNA synthetase, but no evidence of homogeneously staining regions or double minute chromosomes was found. The resistant cell lines should prove useful for the study of the regulation of threonyl-tRNA synthetase. 相似文献