Tryptophan Synthase Activity Research Articles

Dependency on serine concentration of the activity of tryptophan synthase. Cooperative properties.

The activity of the enzyme tryptophan synthase from Escherichia coli was tested as a function of the concentration of L-serine which serves as a substrate in the indole to tryptophan reaction as well as for the L-serine deaminase activity. L-Serine binding was found to follow the pattern of negative cooperativity both by kinetic and by equilibrium methods. The enzyme kinetic data support the view that a rapid equilibration model for the enzyme . substrates complex formation is not strictly obeyed.

Einfluß von Amitrol aufClaviceps fusiformis, Stamm SD 58

The influence of amitrole (3-amino-1,2,4-triazole) on growth, alkaloid formation and on some aromatic as well as ergoline pathway specific enzymes were studied in Claviceps strain, SD 58. In a phosphate-rich medium the addition of amitrole can partially reverse the alkaloid-depressing effect of phosphate. Using a typical fermentation medium amitrole reduces the activities of ergoline specific enzymes but increases drastically tryptophan synthase activity and to a lesser extent transaminase of aromatic amino acids.

Einfluß von Amitrol auf Claviceps fusiformis, Stamm SD 58

AbstractThe influence of amitrole (3‐amino‐1.2,4‐triazole) on growth, alkaloid formation and on some aromatic as well as ergoline pathway specific enzymes were studied in Claviceps strain, SD 58. In a phosphate‐rich medium the addition of amitrole can partially reverse the alkaloid‐depressing effect of phosphate. Using a typical fermentation medium amitrole reduces the activities of ergoline specific enzymes but increases drastically tryptophan synthase activity and to a lesser extent transaminases of aromatic amino acids.

Veränderungen von Tryptophan-Synthase-, Indol-3-essigsäure-Oxidase- und Peroxidase-Aktivität im Verlauf der Entwicklung von Marchantia polymorpha L.

Summary Tryptophan synthase, indole-3-acetic acid oxidase and peroxidase activity could be detected and partly characterised in Marchantia polymorpha . Maximum activity of tryptophan synthase was achieved at pH 7.8 with a protein fraction precipitated between 0-35% ammonium sulfate. Km for L-serine was 3.2 mM. By extraction at various pH-values and by different concentrations of ammonium sulfate we achieved partial fractionation of indole-3-acetic acid oxidase (pH 6.3/35-65%(NH 4 ) 2 SO 4 ) and peroxidase (pH 7.0/35-70%(NH 4 ) 2 SO 4 ). The role of the enzymes in controlling the development of the liverwort is discussed on the basis of their different distribution in the thallus with and without gemma receptacles and the erected fertile branches (antheridiophores and archegoniophores).

Tryptophansynthase in Phycomyces blakesleeanus, Teil II: Tryptophansynthaseaktivität des Pilzes in Abhängigkeit von Lichtbedingungen und vom Zinkgehalt des Kulturmediums

Abstract Tryptophan synthase in Phycomyces blakesleeanus. Part II: Activity of tryptophan synthase in Phycomyces blakesleeanus depending on the light and the content of zinc ions in the culture medium Five‐day‐old cultures of Phycomyces blakesleeanus show notice‐able differences in the phenotype, depending on the culture conditions (permanent light, permanent dark, zinc deficiency, zinc sufficiency) and related to the distribution of tryptophan synthase activity between mycelium and sporangiophores. Permanent light and the presence of zinc ions in the medium during culturing have an antagonistic influence on the tryptophan synthase. The activity of the enzyme is being reduced in the sporangiophores and increased in the mycelium by the influence of light, while zinc ions in the culture medium increase the activity in the sporangiophores at simultaneous reduction in the mycelium.The importance of tryptophan synthase and tryptophan for the development of the fungus in relation to the metabolism of indole acetic acid is discussed.

Tryptophan synthase activity, tryptophan and serine contents in pea (Pisum sativum L.) plants during their ontogenesis

Considerable changes in tryptophan synthase aotivity occur during the ontogenesis of pea plants (Pisum sativum L.) in their individual parts. Maximal tryptophan synthase activity is connected with the vigorous growth period of the individual organs and of the entire plant. The content of free serine which is present in excess and is one of the substrates in the reaction catalyzed by tryptophan synthase, also changes, as well as the content of free tryptophan, the product of the reaction. The changes in the contents of these amino acids do not correspond to the variation in tryptophan synthase activity and mainly follow the alterations in the total nitrogen metabolísm. The limiting factor in the biosynthesis ofL-tryptophanin vivo is probably the availability of the aromatic precursor, above all of indole-3-glycerophosphate. The content of bound tryptophan also shifts in the pea plants owing to the enrichment of proteins in older parts of pea plants with this amino acid.

The influence of Zn2+ ions on the tryptophan biosynthesis in plants

Zn2+ ions slightly enhance at low concentrations (0.01 μg ml-1) the activity of tryptophan synthase obtained from the shoots of 14-day-old pea seedlings (Pisum sativum L.). On the contrary, high concentrations of Zn2+ (10 μg ml-l) exert an inhibitory effect. The direct Zn2+ activation of tryptophan synthase, establishedin vitro with a partially purified enzyme preparation, is relatively low and obviously is not decisive from the point of view of tryptophan biosynthesis of the enzyme and thus they are participating in thein vivo experiments.

Compartmentation of the tryptophan-synthase-proteolyzing system in Saccharomyces cerevisiae.

Ficoll‐gradient fractionation of a metabolic lysate from yeast spheroplasts was used to prepare vacuole, mitochondria and cytosol fractions. Tryptophan synthase activity was exclusively found in the soluble fraction. Proteinases A, B and C as well as the tryptophan‐synthase‐inactivating activity were mainly found in the vacuole fraction. The heat‐stable activities inhibiting pröteinase and tryptophan‐synthase‐inactivating enzyme appeared in the soluble fraction. The subcellular separation of tryptophan synthase and the tryptophan‐synthase‐inactivating activities explains why in crude extracts from stationary yeast cells, high tryptophan synthase activities are observed in spite of high tryptophan‐synthase‐inactivating activity. The proteinases of a freshly prepared vacuole fraction from yeast spheroplasts are unable to inactivate externally added tryptophan synthase unless the organelles are disrupted by sonication or osmotic shock.

Tryptophan synthase from yeast. Purification by affinity chromatography, physical properties.

Tryptophan synthase was purified from Saccharomyces cerevisiae with the aid of affinity chromatography up to a specific activity of 525 units/g protein. Electrophoresis on polyacrylamide gel showed one main protein component. Gel filtration on Sephadex G‐150 indicates an average molecluar weight of 143000. Dissociation with dodecylsulfate followed by dodecyl‐sulfate‐acrylamide gel electrophoresis leads to the conclusion that the enzyme is probably composed of four subunits of equal size. The Km‐values with respect to its substrates indoleglycerol phosphate, indole and l‐serine were estimated. l‐Serine was shown to increase the Km‐value for indoleglycerol phosphate about 7‐fold. Several indole analogues inhibit the tryptophan synthase reaction (B) (i.e. indole + serine → tryptophan); indoleacrylic acid is the most effective. The pH‐optimum of tryptophan synthase is around 6.9 to 7.0. Sulfhydryl modification by 5,5′‐dithiobis(2‐nitrobenzoic acid) and p‐chloromercuriphenylsulfonic acid results in a large loss of tryptophan synthase activity, which cannot be prevented by the substrates l‐serine and indole. The tryptophan synthase inactivating system from yeast affects the catalysis of both reactions (A) (i.e. indoleglycerol phosphate → indole + glyceraldehyde 3‐phosphate) and (B).

Partial purification and properties of tryptophan synthase of pea plants

Tryptophan synthase [ l-serine hydro-lyase (adding indole), EC 4.2.1.20] was extracted and purified approximately 50-fold from pea ( Pisum sativum L. cv. Alaska) seedlings, and found to be a two-component (A and B) enzyme with a molecular weight of approximately 140,000. The enzyme catalyzes three reactions: Indole-3-glycerol phosphate + l-serine → l-tryptophan + glyceraldehyde -3-phosphate + H 2O (1) Indole + l-serine → l-tryptophan + H 2O (2) Indole-3-glycerol phosphate ⇌ indole + glyceraldehyde-3-phosphate (3) Both components of the enzyme are required for Reaction 1, the B component catalyzes Reaction 2, and the A component catalyzes Reaction 3 but only at a fraction of the velocities of Reactions 1 and 2. Reactions 1 and 2 require pyridoxal phosphate. The pH optimum for both Reactions 1 and 2 is approximately 7.6. The enzyme is activated by K + > Na + ∼- NH 4 +, although an absolute univalent cation requirement was not demonstrable. Kinetic constants for Reaction 2 were ascertained for partially purified enzyme extracts. Kinetic data are consistent with an enzyme reaction mechanism involving the formation of a ternary complex. l-Serine may function as an allosteric effector as well as a substrate. The specific activity of tryptophan synthase was determined for cell-free enzyme extracts of different parts of 14-day-old seedlings and was found to be consistently highest in extracts of shoot tips.

The effect of free amino acids and related compounds on the activity of plant enzymes. I. Effect of DL-threonine, L-proline, γ-aminobutyric acid, L-asparagine and L-cysteine on the activity of tryptophan synthase (4.2.1.20) in living bean leaf (Phaseolus vulgaris L.)

The effect of free amino acids and related compounds on the activity of plant enzymes. I. Effect of DL-threonine, L-proline, γ-aminobutyric acid, L-asparagine and L-cysteine on the activity of tryptophan synthase (4.2.1.20) in living bean leaf (Phaseolus vulgaris L.)