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1.
The effect of several cations on the inhibition by PPi of the enzyme myo-inositol-1-phosphate synthase (1L-myo-inositol-1-phosphate lyase (isomerizing) EC 5.5.1.4) from Neurospora crassa was studied. The study wastol biosynthesis can occur in the presence of an intracellular PPi concentration which exceeds the Ki for the enzyme by 350-fold. The inhibition of enzyme activity by PPi,at pH 7.7, was reversed, in decreasing order of effectiveness, by Mn-2, Fe-3 相似文献
2.
The purification of Neurospora crassa myo-inositol-1-phosphate synthase (EC 5.5.1.4) was studied by affinity chromatography using the substrate (glucose-6-phosphate), the inhibitor (pyrophosphate), the coenzyme (NAD+) and the coenzyme analogues (5'AMP and Cibacron Blue F3G-A) of the enzyme as adsorbents attached to agarose gel. Myo-inositol-1-phosphate synthase could be separated completely from the contaminating substance, glucose-6-phosphate dehydrogenase (EC 1.1.1.49), on Blue Sepharose CL-6B and on pyrophosphate-Sepharose. The purified enzyme had a specific activity of 16 400 U/mg. The sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the 60 micrograms of this purified enzyme gave a homogenous band. The enzyme was found to be composed of four identical subunits having a molecular weight of 65 000. 相似文献
3.
J. Aradi A. Zsindely Á. Kiss M. Szabolcs M. Schablik 《Preparative biochemistry & biotechnology》2013,43(2):137-151
The purification of Neurospora crassa myo-inositol-1-phosphate synthase (EC 5.5.1.4) was studied by affinity chromatography using the substrate (glucose-6-phosphate), the inhibitor (pyrophosphate), the coenzyme (NAD+) and the coenzyme analogues (5′AMP and Cibacron Blue F3G-A) of the enzyme as adsorbents attached to agarose gel. Myo-inositol-1-phosphate synthase could be separated completely from the contaminating substance, glucose-6-phosphate dehydrogenase (EC 1.1.1.49), on Blue Sepharose CL-6B and on pyrophosphate-Sepharose. The purified enzyme had a specific activity of 16 400 U/mg. The sodium dodecyl sulfate/polyacrylamide gel electrophoresis of 60 μq of this purified enzyme gave a homogenous band. The enzyme was found to be composed of four identical subunits having a molecular weight of 65 000. 相似文献
4.
Cloned myo-inositol-1-phpsphate synthase (INOS) of Drosophila melanogaster was expressed in Escherichia coli, and purified using a His-affinity column. The purified INOS required NAD+ for the conversion of glucose-6-phosphate to inositol-1-phosphate. The optimum pH for myo-inositol-1-phosphate synthase is 7.5, and the maximum activity was measured at 40 degrees C. The molecular weight of the native enzyme, as determined by gel filtration, was approximately Mr 271,000 +/- 15,000. A single subunit of approximately Mr 62,000 +/- 5,000 was detected upon SDS-polyacrylamide gel electrophoresis. The Michaelis (Km) and dissociation constants for glucose-6-phosphate were 3.5 and 3.7 mM, whereas for the cofactor NAD+ these were 0.42 and 0.4 mM, respectively. 相似文献
5.
The herbicide glyphosate (N-phosphonomethyl glycine) is a potent reversible inhibitor of the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase activity of the purified arom multienzyme complex from Neurospora crassa. Inhibition of the EPSP synthase reaction by glyphosate is competitive with respect to phosphoenolpyruvate, with K(i) 1.1 microM, and uncompetitive with respect to shikimate-3-phosphate. The kinetic patterns are consistent with a compulsory order sequential mechanism in which either PEP or glyphosate can bind to an enzyme: shikimate-3-phosphate complex. 相似文献
6.
A Zsindely M Szabolcs M Kávai M Schablik J Aradi G Szabó 《Acta biologica Academiae Scientiarum Hungaricae》1979,30(1):141-149
Immunological experiments were performed to demonstrate myo-inositol-1-phosphate synthase (EC 5.5.1.4) and its assumed defective variant in various Neurospora crassa stains. An enzymatically inactive protein fraction was isolated from the inl-mutant by the same procedure as that of the enzyme. It consisted of several components by gel electrophoresis, and produced a positive immune reaction demonstrated by immunodiffusion using immune sera produced against the enzyme. Using immunodisc gel electrophoresis it produced an immunoprecipitate of slightly lower mobility than the enzyme itself. Similarly, positive immune reactions were obtained with the enzyme using immune sera produced against the protein fraction isolated from the inl- mutant. Enzyme activity was demonstrated both in a strain transformed by wild-type DNA and in a spontaneous revertant. The enzymes were subsequently isolated from both strains, and some properties were compared with those of the wild-type enzyme. The specific activities were lower but the Michaelis constants were nearly the same. The immunodisc gel electrophoretic patterns of these enzymes were similar to that of the protein fraction from the inositol requiring mutant. 相似文献
7.
E O Ogunyemi F Pittner O Hoffmann-Ostenhof 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1978,359(5):613-616
Using the technique of affinity chromatography on NAD-Sepharose the myo-inositol-1-phosphate synthase of Lemna gibba was purified to homogeneity. The molecular and catalytic properties of this enzyme differ very much from those of myo-inositol-1-phosphate synthase from animal sources. Thus the specific activity of the duckweed enzyme is more than two orders of magnitude higher than that of the enzyme from rat testes. It is inhibitied by EDTA and can be reactivated by Mn2. Its molecular weight (135000 +/- 5000), its subunit composition (3 subunits with identical electrophoretic behaviour) and its isoelectric point (pH 7.7) are also very different from the corresponding parameters for the animal enzyme. 相似文献
8.
Chorismate synthase catalyzes the anti-1,4-elimination of the phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate, a central building block in aromatic amino acid biosynthesis. The enzyme has an absolute requirement for reduced FMN, which in the case of the fungal chorismate synthases is supplied by an intrinsic FMN:NADPH oxidoreductase activity, i.e. these enzymes have an additional catalytic activity. Therefore, these fungal enzymes have been termed "bifunctional." We have cloned chorismate synthase from the common bread mold Neurospora crassa, expressed it heterologously in Escherichia coli, and purified it in a three-step purification procedure to homogeneity. Recombinant N. crassa chorismate synthase has a diaphorase activity, i.e. it catalyzes the reduction of oxidized FMN at the expense of NADPH. Using NADPH as a reductant, a reduced flavin intermediate was observed under single and multiple turnover conditions with spectral features similar to those reported for monofunctional chorismate synthases, thus demonstrating that the intermediate is common to the chorismate synthase-catalyzed reaction. Furthermore, multiple turnover experiments in the presence of oxygen have provided evidence that NADPH binds in or near the substrate (5-enolpyruvylshikimate 3-phosphate) binding site, suggesting that NADPH binding to bifunctional chorismate synthases is embedded in the general protein structure and a special NADPH binding domain is not required to generate the intrinsic oxidoreductase activity. 相似文献
9.
The 3-dehydroquinate synthase activity of the pentafunctional arom enzyme complex of Neurospora crassa is Zn2+-dependent. 总被引:6,自引:5,他引:1
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We have demonstrated the co-purification in constant ratio of all five activities of the pentafunctional arom enzyme complex from Neurospora crassa. Progressive inactivation of the 3-dehydroquinate synthase component of the purified enzyme complex by chelating agents was blocked by the substrate, 3-deoxy-D-arabino-heptulosonate 7-phosphate, but not by the cofactor NAD+. Full activity was restored at Zn2+ concentrations as low as 0.05 nM. Atomic absorption data indicated that the intact enzyme complex contained 1 atom per subunit of tightly bound zinc. The arom 3-dehydroquinate synthase had a calculated turnover number of 19s-1, this being within the narrow range of values obtained for the other four activities of the intact multifunctional enzyme. The Km for 3-deoxy-D-arabino-heptulosonate 7-phosphate was 1.4 microM in a phosphate-free buffer; inorganic phosphate was a competitive inhibitor with respect to 3-deoxy-D-arabino-heptulosonate 7-phosphate. 相似文献
10.
Bornemann S Theoclitou ME Brune M Webb MR Thorneley RN Abell C 《Bioorganic chemistry》2000,28(4):191-204
Chorismate synthase (EC 4.6.1.4) is the shikimate pathway enzyme that catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate. The enzyme reaction is unusual because it involves a trans-1,4 elimination of the C-3 phosphate and the C-6 proR hydrogen and it has an absolute requirement for reduced flavin. Several mechanisms have been proposed to account for the cofactor requirement and stereochemistry of the reaction, including a radical mechanism. This paper describes the synthesis of [4-(2)H]EPSP and the observation of kinetic isotope effects using this substrate with both Neurospora crassa and Escherichia coli chorismate synthases. The magnitude of the effects were (D)(V) = 1.08 +/- 0.01 for the N. crassa enzyme and 1.10 +/- 0.02 on phosphate release under single-turnover conditions for the E. coli enzyme. The effects are best rationalised as substantial secondary beta isotope effects. It is most likely that the C(3)-O bond is cleaved first in a nonconcerted E1 or radical reaction mechanism. Although this study alone cannot rule out a concerted E2-type mechanism, the C(3)-O bond would have to be substantially more broken than the proR C(6)-H bond in a transition state of such a mechanism. Importantly, although the E. coli and N. crassa enzymes have different rate limiting steps, their catalytic mechanisms are most likely to be chemically identical. Copyright 2000 Academic Press. 相似文献
11.
Marcella Schablik Akos Kiss Attila Zsindley Gábor Szabó 《Molecular & general genetics : MGG》1988,213(1):140-143
Summary Slow-growing (inl
+/-) spontaneous mutants have been isolated from an inositol requiring (inl) strain of Neurospora crassa that produces defective myo-inositol-1-phosphate synthase (MIPS), the enzyme responsible for the production of inositol-1-phosphate from glucose-6-phosphate. The defective enzyme has some residual activity. In the inl
+/- strain the synthesis of the defective enzyme is enhanced, which enables the strain to grow slowly on minimal medium. The mutation (opi1) responsible for the partial inositol independence segregates independently from the inositol locus, and suppresses the inositolless character by overproduction of defective MIPS. opi1 acting upon the wild type (inl
+) allele increases MIPS production and causes inositol excretion. 相似文献
12.
Some kinetic properties of the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Neurospora crassa. 总被引:4,自引:1,他引:3
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The steady-state kinetic properties of purified tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Neurospora crassa were examined. The results suggest that the enzyme obeys a Rapid-Equilibrium Ordered mechanism, in which phosphoenolpyruvate is the first substrate to bind and 3-deoxy-D-arabino-heptulosonate 7-phosphate is the second product to be released, rather than a Ping Pong mechanism as has been reported previously. The inhibition by tryptophan was found to be parabolic competitive with respect to D-erythrose 4-phosphate and parabolic non-competitive with respect to phosphoenolpyruvate. The enzyme was inactivated by EDTA, and could be protected against this inactivation by phosphoenolpyruvate or 3-deoxy-D-arabino-heptulosonate 7-phosphate but not by D-erythrose 4-phosphate, tryptophan or Pi. This suggests that the enzyme may be a metalloenzyme. 相似文献
13.
Soboren, Josephine (University of California, Los Angeles), and Joseph F. Nyc. Amino acid interactions in Neurospora crassa. J. Bacteriol. 82:20-25. 1961.-A systematic study of the effects of the naturally occurring amino acids on the growth of a wild-type strain of Neurospora crassa focused attention upon l-tryptophan, which exhibits a strong growth inhibitory effect. Further investigation disclosed that other tryptophan metabolites, anthranilic acid, indole, kynurenine, and 3-hydroxykynurenine also inhibit growth. The proposed antimetabolic role of these aromatic compounds explains the poor growth response of certain tryptophan-requiring strains of N. crassa to tryptophan supplements. The growth of normal and mutant strains of N. crassa on media supplemented with tryptophan is influenced by the presence of other amino acids. 相似文献
14.
15.
Four natural protease inhibitors have been partially purified by heat treatment, ion-exchange chromatography pand gel filtration from Neurospora crassa. The inhibitory activity has been estimated by measuring the inhibition of proteolysis of casein as well as by the protection of Neurospora tryptophan synthase from proteolytic inactivation. The inhibitors are all oligopeptides and possess molecular weights in the range 5000-24 000 and appear to be very specific to Neurospora proteases. They may be classified into two types. The first are specific to Neurospora alkaline protease and the second to acidic protease. None of them exhibited any effect on other proteases including trypsin, chymotrypsin, papain, pepsin, thermolysin, subtilisin and proteinase K. The possible physiological role of these inhibitors is discussed. 相似文献
16.
Genetic and biochemical identification of the glutamate synthase structural gene in Neurospora crassa. 总被引:2,自引:1,他引:1
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Neurospora crassa cells require glutamate synthase activity for growth under ammonium-limiting conditions. Despite the physiological importance of glutamate synthase, little is known about the genetics of its expression. To identify the glutamate synthase structural gene, we isolated three new mutants lacking this activity. All mutations are recessive to the wild-type allele and belong to the same complementation group as the previously described en(am)-2 (C24) mutation. Two lines of evidence indicate that en(am)-2 is the structural gene for glutamate synthase in N. crassa. The en(am)-2+ gene shows a gene dosage effect on enzyme activity, and some mutants lacking glutamate synthase activity have cross-reacting material. These data suggest that the mutations are located in the structural gene for N. crassa glutamate synthase. 相似文献
17.
Immunohistochemical Staining and Enzyme Activity Measurements Show myo-Inositol-1-Phosphate Synthase to Be Localized in the Vasculature of Brain 总被引:1,自引:3,他引:1
Yun-Hua H. Wong Sandra J. Kalmbach Boyd K. Hartman William R. Sherman 《Journal of neurochemistry》1987,48(5):1434-1442
Rabbit anti-bovine myo-inositol-1-phosphate synthase was used to examine the distribution of that enzyme in perfused and immersion-fixed bovine brain and testis. In brain, intense and specific staining was found in the walls of all the vascular elements including cerebral capillaries. The remainder of brain parenchyma exhibited only low levels of background staining. In testis, an organ rich in the enzyme, blood vessels showed no specific staining. Instead, the enzyme was found in the seminiferous epithelium of the seminiferous tubules, perhaps localized in spermatozoa. To confirm the brain finding, the activity of myo-inositol-1-phosphate synthase was measured in bovine brain microvessel preparations and brain pial vessels. In these preparations the activity of the enzyme was found on average to be 7 and 22 times enriched over that in whole brain, respectively. The activities of two other enzymes of inositol metabolism, myo-inosose reductase and myo-inositol-1-phosphatase, were also examined for their distribution in brain. Those enzymes were found to be generally distributed. The surprising finding of a vascular localization of myo-inositol-1-phosphate synthase in brain raises new questions about the mechanism by which myo-inositol is concentrated to such high cellular levels in the principal substance of that organ. 相似文献
18.
1. A morphological mutant of Neurospora crassa, smco 9, (R2508) that exhibits colonial morphology when grown on sucrose or on maltose, showed a partial reversal of this morphology toward that of the wild type when it was grown on potato starch or on isomaltose. 2. A common feature of both potato starch and isomaltose is the presence of alpha-1, 6 glucosidic linkages. This suggested that these morphological effects might be due to differences in alpha-1,4 glucan: alpha-1,4 glucan 6 glycosyltransferase, (EC 2.4.1.18) commonly known as "the branching enzyme". 3. The branching enzyme was purified from wild type, Neurospora crassa, and from the semicolonial mutant, R2508, both grown on sucrose or on potato starch. It has a molecular weight of 140,000 as estimated by gel filtration on a Bio Gel A 1.5 m column. This enzyme plus phosphorylase a in an unprimed reaction catalyzes the synthesis of a branched polysaccharide in vitro. 4. No branching enzyme activity was apparent in extracts of the mutant R2508, grown on potato starch until a thermolabile inhibitor was removed by fractionation on a DEAE column. 5. This inhibitor has a molecular weight greater than 100,000 as estimated on a P-100 polyacrylamide gel column. The specificity of the inhibitor is not absolute in that it inhibits glycogen synthetase in addition to the branching enzyme in Neurospora. 相似文献
19.
F Pittner O Hoffmann-Ostenhof 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1976,357(12):1667-1671
The animal myo-inositol-1-phosphate synthase is competitively inhibited by pyridoxal phosphate and trinitrobenzensulphonate, both compounds known to prevent Schiff's base formation. When incubated with labelled substrate and then treated with NaBH4, label can be recovered in the enzyme protein. In analogous experiments with tritiated NaBH4 the enzyme protein also becomes labelled; after hydrolysis of such protein only one labelled compound, derived from lysine and D-glucose 6-phosphate, could be isolated. Its exact structure is not yet known. From these results it can be concluded that during its action myo-inositol-1-phosphate synthase forms a Schiff's base with its substrate, in analogy to the class I aldolases. 相似文献