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1.
The enzyme threonine deaminase (TD) is a key regulatory enzyme in the pathway for the biosynthesis of isoleucine. TD is inhibited by its end product, isoleucine, and this effect is countered by valine, the product of a competing biosynthetic pathway. Sequence and structure analyses have revealed that the protomers of many TDs have C-terminal regulatory domains, composed of two ACT-like subdomains, which bind isoleucine and valine, while others have regulatory domains of approximately half the length, composed of only a single ACT-like domain. The regulatory responses of TDs from both long and short sequence varieties appear to have many similarities, but there are significant differences. We describe here the allosteric properties of Bacillus subtilis TD ( bsTD), which belongs to the short variety of TD sequences. We also examine the effects of several mutations in the regulatory domain on the kinetics of the enzyme and its response to effectors. The behavior of bsTD can be analyzed and rationalized using a modified Monod-Wyman-Changeux model. This analysis suggests that isoleucine is a negative effector, and valine is a very weak positive effector, but that at high concentrations valine inhibits activity by competing with threonine for binding to the active site. The behavior of bsTD is contrasted with the allosteric behavior reported for TDs from Escherichia coli and Arabidopsis thaliana, TDs with two subdomains. We suggest a possible evolutionary pathway to the more complex regulatory effects of valine on the activity of TDs of the long sequence variety, e.g., E. coli TD.  相似文献   

2.
Isoleucine-deficient mutants of Salmonella typhimurium were isolated. Three groups of mutants can be discerned by their nutritional requirements and enzyme patterns. (i) Mutants which grow with isoleucine alone are devoid of biosynthetic threonine deaminase (TD). (ii) Mutants growing with isoleucine and valine are devoid of transaminase B. (iii) Mutants growing with either isoleucine or threonine have normal levels of TD. However, the sensitivity of this enzyme to feedback inhibition by isoleucine is greatly enhanced. The inhibitory effect of isoleucine can be counterbalanced by high concentrations of threonine. These results indicate that the production of isoleucine in the mutants is restricted to a low level not sufficient to support the growth of the cells. This hypothesis is confirmed by studies with revertants of an isoleucine-threonine mutant. In nine revertants, wild-type properties of TD have been restored. In four revertants, the hypersensitivity of TD is unchanged, but the strains produce a greatly enhanced quantity of threonine, which is excreted into the culture medium. It follows, that hypersensitivity of TD to inhibition by isoleucine is the cause of the nutritional requirement of isoleucine-threonine mutants.  相似文献   

3.
B Mckel  L Eggeling    H Sahm 《Journal of bacteriology》1992,174(24):8065-8072
Threonine dehydratase activity is an important element in the flux control of isoleucine biosynthesis. The enzyme of Corynebacterium glutamicum demonstrates a marked sigmoidal dependence of initial velocity on the threonine concentration, a dependence that is consistent with substrate-promoted conversion of the enzyme from a low-activity to a high-activity conformation. In the presence of the negative allosteric effector isoleucine, the K0.5 increased from 21 to 78 mM and the cooperativity, as expressed by the Hill coefficient increased from 2.4 to 3.7. Valine promoted opposite effects: the K0.5 was reduced to 12 mM, and the enzyme exhibited almost no cooperativity. Sequence determination of the C. glutamicum gene for this enzyme revealed an open reading frame coding for a polypeptide of 436 amino acids. From this information and the molecular weight determination of the native enzyme, it follows that the dehydratase is a tetramer with a total mass of 186,396 daltons. Comparison of the deduced polypeptide sequence with the sequences of known threonine dehydratases revealed surprising differences from the C. glutamicum enzyme in the carboxy-terminal portion. This portion is greatly reduced in size, and a large gap of 95 amino acids must be introduced to achieve homology. Therefore, the C. glutamicum enzyme must be considered a small variant of threonine dehydratase that is typically controlled by isoleucine and valine but has an altered structure reflecting a topological difference in the portion of the protein most likely to be important for allosteric regulation.  相似文献   

4.
Biosynthetic threonine deaminase (TD) from Schizosaccharomyces pombe has been partially purified from crude extracts by treatment with protamine sulfate, ammonium sulfate precipitation, and gel filtration through Sephadex G-25. In both crude extracts and purified preparations, TD showed marked stimulation by pyridoxal phosphate. A pH optimum for activity was found at pH 9.0, whereas the inhibition caused by the natural feedback inhibitor, l-isoleucine, was maximal at pH 7.4. l-Threonine exhibits homotropic cooperative effects at low pH (7.0-8.0), which are eliminated at pH 9.0, and the affinity for substrate (in terms of K(m)) increased with increasing pH. Enzyme activity could be completely inhibited by isoleucine over a pH range of 7.4 to 9.0; the amount of isoleucine required for 50% inhibition increased with increasing pH. Isoleucine inhibition was pseudocompetitive with respect to substrate and increased the cooperative effects of threonine. l-Valine was found to reverse isoleucine inhibition; it also activated the enzyme in a pH range of 7.0 to 8.0 by eliminating the cooperative effects of threonine, thus normalizing the substrate saturation curves at these pH values. l-Leucine was shown to be a competitive inhibitor with respect to threonine, and to be able partially to reverse isoleucine inhibition. Treatment of TD with mercurials did not result in desensitization to isoleucine inhibition. However, at pH 10, virtually no sensitivity of the enzyme to isoleucine was observed while activity remained strong, which suggests the existence of separate sites on the TD molecule for binding threonine and isoleucine. A tentative model is presented which unifies the kinetic results reported here in terms of the interactions of TD with its effector molecules.  相似文献   

5.
Induced Phenotypic Resistance to Valine in Mycobacterium pellegrino   总被引:1,自引:0,他引:1       下载免费PDF全文
Valine coordinately increases the levels of three of the enzymes participating in the biosynthesis of isoleucine and valine in Mycobacterium pellegrino. The amount of valine required for end-product induction depends on the condition of the cells. Isoleucine inhibits the effect of valine. Acetohydroxy acid synthetase, the enzyme catalyzing the first common step in the biosynthesis of valine and isoleucine, is inhibited by valine. The induction effect of valine appears to be due to its ability to inhibit the activity of this enzyme, thus causing isoleucine deficiency, which in turn leads to derepression. This conclusion is supported by the fact that valine, under certain conditions, inhibits growth.  相似文献   

6.
Threonine dehydratase (TD; EC.4.2.1.16) is a key enzyme involved in the biosynthesis of isoleucine. Inhibition of TD by isoleucine regulates the flow of carbon to isoleucine. We have identified two different forms of TD in tomato (Lycopersicon esculentum) leaves. One form, present predominantly in younger leaves, is inhibited by isoleucine. The other form of TD, present primarily in older leaves, is insensitive to inhibition by isoleucine. Expression of the latter enzyme increases as the leaf ages and the highest enzyme activity is present in the old, chlorotic leaves. The specific activity of the enzyme present in older leaves is much higher than the one present in younger leaves. Both forms can use threonine and serine as substrates. Whereas TD from the older leaves had the same Km (0.25 mM) for both substrates, the enzyme from the young leaves preferred threonine (Km = 0.25 mM) over serine (Km = 1.7 mM). The molecular masses of TD from the young and the old leaves were 370,000 and 200,000 D, respectively. High levels of the isoleucine-insensitive form of threonine dehydratase in the older leaves suggests an important role of threonine dehydratase in nitrogen remobilization in senescing leaves.  相似文献   

7.
The regulatory properties of acetohydroxy acid synthetase (AHAS), the first enzyme in the biosynthetic pathway to valine and the second in the isoleucine pathway, were investigated in the fission yeast Schizosaccharomyces pombe. The enzyme was partially purified from crude extracts by protamine sulfate treatment, ammonium sulfate fractionation, and gel filtration through Sephadex G-25. AHAS from S. pombe is unique in that its activity shows a single peak around pH 6.5; high sensitivity to feedback inhibition by valine at this pH (K(i) = 0.1 mM) indicates that the enzyme is involved in valine biosynthesis. Pyruvate saturation kinetics of AHAS extracted from cells grown on glycerol as sole carbon and energy source were normal and hyperbolic. In contrast, the enzyme from glucose-grown cells exhibited sigmoidal saturation kinetics, an effect which disappeared when the synthetase from such cells was partially purified. This phenomenon was shown to be due to competition for pyruvate between AHAS and pyruvate decarboxylase; the latter enzyme is present in large amounts in cells fermenting glucose. Valine inhibition is noncompetitive in nature, and this effector exhibits homotropic cooperative effects; isoleucine is a less-potent inhibitor of AHAS activity. Mercurial treatment reversibly desensitized the enzyme to valine inhibition. On the basis of these data, the S. pombe AHAS appears to be an allosteric regulatory enzyme with the properties of a negative V system.  相似文献   

8.
A spontaneous leu-linked mutation (ilvH2015) in Escherichia coli K-12 made the strain resistant to 1 mM valine and l mM glycylvaline (Val-r) and caused the isoleucine and valine biosynthetic enzyme, acetohydroxy acid synthase, to be less sensitive to feedback inhibition by valine than the wild-type enzyme. Transfer of the ilvDAC deletion into a strain carrying ilvH2015 abolished the effect of the marker on the acetohydroxy acid synthase and rendered it as sensitive to valine as the enzyme in the isogenic control strain without the Val-r marker under both repressing and limiting conditions. In contrast, auxotrophy caused by transfer of an ilvC lesion into the Val-r strain did not interfere with the effect of ilvH2015 on valine sensitivity of acetohydroxy acid synthase. In addition, the presence of the Val-r marker produced minor but significant pleiotropic effects on several other isoleucine and valine biosynthetic enzymes but did not cause derepression of the ilv gene cluster. These studies suggest some type of interaction between a product produced by a gene close to leu and the isoleucine and valine biosynthetic enzymes.  相似文献   

9.
Threonine deaminase [EC 4.2.1.16] was highly purified from Bacillus stearothermophilus. The enzyme exhibited maximum activity at 65 degrees and at pH 9.2--9.6. It was inactivated on dilution and on storage at 4 degrees, but was protected by egg albumin. The enzyme was labile at 65 degrees, but became stable in the presence of egg albumin and isoleucine at pH 7.0. The substrate saturation curve for the enzyme reaction at 40 or 65 degrees was hyperbolic, but in the presence of isoleucine, the curve became sigmoidal (n = 2). The enzyme was more sensitive to isoleucine at 40 degrees than at 65 degrees, while valine slightly inhibited the enzyme at both 40 and 65 degrees. Inhibition of the enzyme by isoleucine was antagonized by valine at 40 and 65 degrees. These properties were essentially similar to those of the enzymes from mesophilic and thermophilic bacteria. The enzyme existed in two forms with different molecular sizes, 1.5-5 X 10(6) and 2 X 10(5) daltons, at pH 7.0 and at temperatures below 40 degrees. The larger component disaggregated into the small one at pH 8.5 or above, at temperatures above 50 degrees or in the presence of isoleucine and valine.  相似文献   

10.
a-Isopropylmalate synthase activity was demonstrated in the Sephadex G 25 gel filtrated crude extracts of one yeast and 43 bacterial strains belonging to 14 families. The enzyme was inhibited by leucine from all strains Bacteroides fragilis, Clostridia and several phototropic bacteria. The enzyme was inhibited by leucine from all strains investigated. In crude extracts of 17 species (8 genera) the leucine-mediated inhibition could be relieved by the addition of valine or isoleucine , but not by the addition of threonine or alanine. The enzymes from 11 species (7 genera) were inhibited by 1 mM valine and isoleucine, whereas the enzyme activity from 5 bacteria (4genera) were not so affected. These results suggest that valine and isoleucine are specifically involved in the regulation of leucine biosynthesis in several bacteria. The affect of valine and isoleucine on the IPM-synthase activity from mycobacteria and Corynebacterium autotrophicum lends support to the reclassification of Mycobacterium flavum 301 to C. autotrophicum. The antagonism between 5′,5′,5′-trifluoroleucine and amino acids and a-ketoisovalerate was a-isopropylmalate synthase in the presence or abssence of leucine and the reversal of the 5′,5′,5′-trifluoroleucine-mediated growth inhibition by these amino acids.  相似文献   

11.
High quality purification of membrane-spanning peptides and proteins remains a challenging problem. In this work we describe a tailored chromatographic purification of a synthetic 35-residue peptide corresponding to the transmembrane region of the tyrosine kinase receptor c-erb2/neu. Composed to over 70% by the amino acids alanine, isoleucine, leucine, phenylalanine and valine, this peptide presents a very hydrophobic character. Product isolation from the complex peptide mixture, obtained after acid cleavage of the resin matrix used during the solid-phase synthesis, represents a difficult task. We propose a three step strategy based on gel permeation and reversed-phase high-performance liquid chromatography, using aprotic polar solvent mixtures. The challenge consisted in obtaining a sufficient amount of an extremely pure sample, in order to allow structural analysis by NMR spectroscopy. Keeping trace of the synthetic peptide throughout the different purification steps was assured by MALDI TOF mass spectrometry, and the final product purity was checked by coupled liquid chromatography–ESI TOF mass spectrometry.  相似文献   

12.
In a strain of Escherichia coli K-12 lacking threonine deaminase, the enzyme converting alpha-ketoisovalerate and alpha-keto-beta-methylvalerate to valine and isoleucine, respectively, was multivalently repressed by valine, isoleucine, and leucine. This activity was due to transaminase B, specified by the ilvE structural gene.  相似文献   

13.
The synthesis of the three types of acetolactate synthase (EC 4.1.3.18) which are responsible for the biosynthesis os isoleucine and valine, was observed in Aerobacter aerogenes I-12, an isoleucine-requiring mutant, when grown on the four kinds of media. When the cells were grown on isoleucine-rich medium, acetolactate synthase sensitive to feedback inhibition and having an optimum pH at 8.0 was formed. By increasing the amount of potassium phosphate in the medium, the catabolite repression of the enzyme having an optimum pH at 6.0 and which is insensitive to feedback inhibition, was released. In contrast, acetolactate synthase having an optimum pH at 8.0 and insensitive to feedback inhibition was formd when isoleucine was limited, irrespective of phosphate concentrations. Two insensitive enzymes were not regulated by isoleucine, leucine and valine, although sensitive pH 8.0 enzyme was repressed by them. Thus, it may be assumed that the synthesis of insensitive pH 8.0 enzyme were repressed by limiting the amount of isoleucine is still open.  相似文献   

14.
Mourad G  King J 《Plant physiology》1995,107(1):43-52
Threonine dehydratase/deaminase (TD), the first enzyme in the isoleucine biosynthetic pathway, is feedback inhibited by isoleucine. By screening M2 populations of ethyl methane sulfonate-treated Arabidopsis thaliana Columbia wild-type seeds, we isolated five independent mutants that were resistant to L-O-methylthreonine, an isoleucine structural analog. Growth in the mutants was 50- to 600-fold more resistant to L-O-methylthreonine than in the wild type. The resistance was due to a single, dominant nuclear gene that was denoted omr1 and was mapped to chromosome 3 in GM11b, the mutant line exhibiting the highest level of resistance. Biochemical characteristics (specific activities, Km, Vmax, and pH optimum) of TD in extracts from the wild type and GM11b were similar except for the inhibition constant of isoleucine, which was 50-fold higher in GM11b than in the wild type. Levels of free isoleucine were 20-fold higher in extracts from GM11b than in extracts from wild type. Therefore, isoleucine feedback insensitivity in GM11b is due to a mutant form of the TD enzyme encoded by omr1. The mutant allele omr1 of the line GM11b could provide a new selectable marker for plant genetic transformation.  相似文献   

15.
Dual functional roles of ATP in the human mitochondrial malic enzyme   总被引:2,自引:0,他引:2  
Hsu WC  Hung HC  Tong L  Chang GG 《Biochemistry》2004,43(23):7382-7390
Human mitochondrial malic enzyme is a regulatory enzyme with ATP as an inhibitor. Structural studies reveal that the enzyme has two ATP-binding sites, one at the NAD(+)-binding site in the active center and the other at the exo site in the tetramer interface. Inhibition of the enzyme activity is due to the competition between ATP and NAD(+) for the nucleotide-binding site at the active center with an inhibition constant of 81 microM. Binding of the ATP molecule at the exo site, on the other hand, is important for the maintenance of the quaternary structural integrity. The enzyme exists in solution at neutral pH and at equilibrium of the dimer and tetramer with a dissociation constant (K(TD)) of 0.67 microM. ATP, at a physiological concentration, shifts the equilibrium toward tetramer and decreases the K(TD) by many orders of magnitude. Mutation of a single residue Arg542 at the tetrameric interfacial exo site resulted in dimeric mutants. ATP thus has dual functional roles in the mitochondrial malic enzyme.  相似文献   

16.
Concerted efforts were directed towards understanding the control of acetohydroxy acid synthase (AHAS) in the gyrB mutant hisU1820 of Salmonella typhimurium. A media shift from valine to valine plus isoleucine causes a dramatic 4 to 5 fold burst of AHAS valine sensitive activity which appears to be dependent on translation. DJ19, an isolated valine sensitive derivative of the gyrB mutant, maintains a dramatic increase in AHAS valine sensitive activity upon the addition of isoleucine to valine supplemented cultures, suggesting that the isoleucine effect is specific for valine sensitive AHAS. Evidence supports isoleucine as a positive effector on valine sensitive AHAS expression and that the gyrB mutation accentuates the isoleucine effect.  相似文献   

17.
The metabolism of isoleucine to active amyl alcohol (2-methylbutanol) in yeast was examined by the use of (13)C nuclear magnetic resonance spectroscopy, combined gas chromatography-mass spectrometry, and a variety of mutants. From the identified metabolites a number of routes between isoleucine and active amyl alcohol seemed possible. All involved the initial decarboxylation of isoleucine to alpha-keto-beta-methylvalerate. The first, via branched chain alpha-ketoacid dehydrogenase to alpha-methylbutyryl-CoA, was eliminated because abolition of branched-chain alpha-ketoacid dehydrogenase in an lpd1 disruption mutant did not prevent the formation of active amyl alcohol. However, the lpd1 mutant still produced large amounts of alpha-methylbutyrate which initially seemed contradictory because it had been assumed that alpha-methylbutyrate was derived from alpha-methylbutyryl-CoA via acyl-CoA hydrolase. Subsequently it was observed that alpha-methylbutyrate arises from the non-enzymic oxidation of alpha-methylbutyraldehyde (the immediate decarboxylation product of alpha-keto-beta-methylvalerate). Mutant studies showed that one of the decarboxylases encoded by PDC1, PDC5, PDC6, YDL080c, or YDR380w must be present to allow yeast to utilize alpha-keto-beta-methylvalerate. Apparently, any one of this family of decarboxylases is sufficient to allow the catabolism of isoleucine to active amyl alcohol. This is the first demonstration of a role for the gene product of YDR380w, and it also shows that the decarboxylation steps for each alpha-keto acid in the catabolic pathways of leucine, valine, and isoleucine are accomplished in subtly different ways. In leucine catabolism, the enzyme encoded by YDL080c is solely responsible for the decarboxylation of alpha-ketoisocaproate, whereas in valine catabolism any one of the isozymes of pyruvate decarboxylase will decarboxylate alpha-ketoisovalerate.  相似文献   

18.
Liver arginase of Clarias batrachus has been purified to 56.3-fold employing ammonium sulphate fraction, DEAE-cellulose and CM-cellulose chromatography. Bidirectional polyacrylamide gel electrophoresis shows the presence of two isoenzymes of arginase. The enzyme has a molecular weight of about 87,000 and Km 15.38 mM for L-arginine, optimum pH 9.5 and temperature 37 degrees C. Ornithine and leucine as competitive whereas valine and isoleucine act as non-competitive inhibitors with respect to L-arginine as substrate.  相似文献   

19.
A Nicotiana plumbaginifolia cell line able to grow in the presence of high doses of valine was isolated following -rays mutagenesis. The selected clone, named D5R5, showed a growth rate higher than that of wild-type. It was less sensitive also to an equimolar mixture of the three branched-chain amino acids, but did not display cross-resistance to isoleucine and leucine. The increased tolerance was due to neither a reduced valine uptake, nor a modification in the level or sensitivity to feed-back inhibition by valine of the first common enzyme (and the main regulative site) in isoleucine, leucine and valine synthesis, acetohydroxyacid synthase (AHAS). When wild-type cells were fed with valine or equimolar mixtures of the three aminoacids, a decrease in AHAS level was found. On the contrary, the level of extractable AHAS activity from D5R5 cells was significantly less affected by similar treatments, suggesting that some alteration in enzyme modulation mechanism(s) could account for valine resistance.Abbreviations AHAS acetohydroxyacid synthase - BCAA branched-chain amino acid - FAD flavin adenine dinucleotide - ILV equimolar mixture of isoleucine, leucine and valine - TPP thiamine pyrophosphate  相似文献   

20.
Methylmalonate semialdehyde dehydrogenase was purified from rat liver in order to define the distal portion of valine catabolism and related pathways in mammals. The purified enzyme is active with malonate semialdehyde and consumes both stereoisomers of methylmalonate semialdehyde, implicating a single semialdehyde dehydrogenase in the catabolism of valine, thymine, and compounds catabolized by way of beta-alanine. The oxidation of malonate and methylmalonate semialdehydes by this enzyme is CoA-dependent, the products being acetyl-CoA and propionyl-CoA, respectively. Expected activity with ethylmalonate semialdehyde as substrate was not found. Methylmalonate semialdehyde dehydrogenase was separated on DEAE-Sephacel into two isoforms which differ in mobility during nondenaturing polyacrylamide gel electrophoresis. The two forms are immunologically cross-reactive and exhibit the same N-terminal sequence, suggesting that one form is the product of the other. The monomer molecular mass, determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was 58 kDa. The native molecular mass, estimated by gel filtration, was 250 kDa, suggesting a tetrameric structure.  相似文献   

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