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排序方式: 共有233条查询结果,搜索用时 46 毫秒
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Yoav Peleg Emil Battat Michael C. Scrutton Israel Goldberg 《Applied microbiology and biotechnology》1989,32(3):334-339
Summary Electrophoretic studies of fumarase and nicotine adenine dinucleotide (NAD)-malate dehydrogenase were carried out in the fumaric acid-accumulating fungus Rhizopus oryzae. The analyses revealed two fumarase isoenzymes, one localised solely in the cytosol and the other found both in the cytosol and in the mitochondrial fraction. The activity of the cytosolic isoenzyme of fumarase was higher during the acid production stage than during growth. Addition of cycloheximide inhibited fumaric acid production and decreased the activity of the cytosolic isoenzyme of fumarase. These results suggested that de novo protein synthesis is required for increase in the activity of the cytosolic isoenzyme and that such an increase in activity is essential for fumaric acid accumulation. Three distinct isoenzymes of NAD-malate dehydrogenase could be detected in R. oryzae. No changes were observed in the isoenzyme pattern of malate dehydrogenase during fumaric acid production. 相似文献
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M P Deonarain N S Scrutton A Berry R N Perham 《Proceedings. Biological sciences / The Royal Society》1990,241(1302):179-186
Directed mutagenesis of the gor gene from Escherichia coli encoding the flavoprotein glutathione reductase was used to convert the two cysteine residues that comprise its redox-active disulphide bridge to alanine (C42A) and serine (C47S) residues. A double mutant (C42AH439A) was also created in which His-439, the proton donor/acceptor in the glutathione-binding site, was additionally converted into an alanine residue. The C42A and C47S mutants were both unable to catalyse the reduction of glutathione by NADPH. The C42A mutant retained the transhydrogenase activity of the wild-type enzyme, whereas the C47S mutant was also inhibited in this reaction. These results support the view that in the catalytic mechanism of E. coli glutathione reductase, the thiolate form of Cys-42 acts as a nucleophile to initiate disulphide exchange with enzyme-bound glutathione and that the thiolate form of Cys-47 generates an essential charge-transfer complex with enzyme-bound FAD. Titration of the C42A and C42AH439A mutants indicated that the imidazole side-chain of His-439 lowered the pKa of the charge-transfer thiol (Cys-47) from 7.7 to 5.7, enhancing its ability to act as an anion at neutral pH. Several important differences between these mutants of E. coli glutathione reductase and similar mutants (or chemically modified forms) of other members of the flavoprotein disulphide oxidoreductase family were noted, but these could be explained in terms of the different redox chemistries of the enzymes concerned. 相似文献
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Addition of GTP markedly enhances the ability of thrombin to cause a leftward shift in the Ca2+ dose/response curve for 5-hydroxytryptamine secretion from permeabilised human platelets. Little effect is observed on addition of GTP in the absence of thrombin. Neither ADP nor adrenaline, in the presence or absence of GTP, causes such a shift, whereas 5-hydroxytryptamine does so to a small extent but only in the presence of GTP. The leftward shift in the Ca2+ dose/response curve induced by 12-O-tetradecanoyl-phorbol-13-acetate or 1-oleyl-2-acetylglycerol is not enhanced by addition of GTP. The thrombin concentration required for half-maximal enhancement of the response to Ca2+ is markedly reduced by addition of GTP. The results support the postulate that the effects of excitatory agonists in this system correlate with their ability to activate phospholipase C and provide further evidence for a role for GTP in signal transduction between the receptor and phospholipase C. 相似文献
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Two gor genes encoding different mutants of Escherichia coli glutathione reductase have been expressed in the same E. coli cell, leading to the creation of a hybrid form of the enzyme dimer. One of the gor genes carried, in addition to various directed mutations, a 5' extension that encodes a benign penta-arginine "arm" added to the N-terminus of the glutathione reductase polypeptide chain [Deonarain, M.P., Scrutton, N.S., & Perham, R.N. (1992) Biochemistry (preceding paper in this issue)]. This made possible, by means of ion-exchange chromatography or nondenaturing polyacrylamide gel electrophoresis, the facile separation of the hybrid enzyme from the two parental forms. Moreover, the two subunits in the hybrid enzyme could be made to carry different mutations. In this way, glutathione reductases with only one active site per dimer were generated: the effects of replacing tyrosine-177 with glycine in the NADPH-binding site, which greatly diminishes the Km for glutathione and switches the kinetic mechanism from ping-pong to ordered sequential, and of replacing His-439 with glutamine in the glutathione-binding site, which greatly diminishes the Km for NADPH, were both found to be restricted to the one active site carrying the mutations. This system of generating separable enzyme hybrids is generally applicable and should make it possible now to undertake a more systematic study of catalytic mechanism and assembly for the many enzymes with quaternary structure. 相似文献
9.
Protein recognition of ammonium cations using side-chain aromatics: a structural variation for secondary ammonium ligands.
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A. R. Raine C. C. Yang L. C. Packman S. A. White F. S. Mathews N. S. Scrutton 《Protein science : a publication of the Protein Society》1995,4(12):2625-2628
A model for the structure of dimethylamine dehydrogenase was generated using the crystal coordinates of trimethylamine dehydrogenase. Substrate is bound in trimethylamine dehydrogenase by cation-pi bonding, but modeling of dimethylamine dehydrogenase suggests that secondary amines are bound by a mixture of cation-pi and conventional hydrogen bonding. In dimethylamine dehydrogenase, binding is orientationally more specific and distinct from those proteins that bind tertiary and quaternary amine groups. 相似文献
10.
Pyruvate carboxylase from Aspergillus nidulans. Effects of regulatory modifiers on the structure of the enzyme 总被引:12,自引:0,他引:12
S A Osmani F Mayer F A Marston I P Selmes M C Scrutton 《European journal of biochemistry》1984,139(3):509-518
Pigment-binding protein of the facultatively phototrophic bacterium Rhodospeudomonas capsulata could be selectively synthesized in toluene-treated cells as well as in homologous and heterologous cell-free translation systems by isolated polysomes. It is shown that the pigment-binding polypeptides of the light-harvesting complexes are encoded by messenger RNA of extreme longevity. The dependence of their synthesis on the concomitant synthesis of tetrapyrroles was demonstrated in the toluene-treated cells. The large Mr-28 000 polypeptide of the reaction center and the Mr-10 000 pigment-binding polypeptide of the light-harvesting complex II were found to be synthesized by free (water-soluble) polysomes without a cleavable 'leader' or 'signal' peptide [reviewed by W. Wickner (1979) Annu. Rev. Biochem. 48, 23-45]. The Mr-10 000 polypeptide, as synthesized in vitro, was studied in more detail. Unlike the membrane-assembled polypeptide in vivo it was insoluble in an organic solvent mixture (chloroform/methanol 1:1, v/v). After detergent denaturation in the presence of membrane isolated from the organism it became organic-solvent-soluble. Obviously the polypeptide could be induced to assume alternative conformations in which its apolar residues were either exposed to the solvent or buried within. These findings, in agreement with Wickner's hypothesis, indicate that the Mr-10 000 polypeptide may enter the lipid bilayer by a 'membrane-triggered' conformational change. 相似文献