[Serine and threonine dehydratases in representatives of the Enterobacteriaceae family].
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Enterobacteriaceae↗
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Results for “Threonine Dehydratase”
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Control of biodegradative threonine dehydratase inducibility by cyclic AMP in energy-restricted Escherichia coli.
To explain the requirement for anaerobic conditions in the induction of biodegradative L-threonine dehydratase in Escherichia coli, Crookes strain, measurements of cyclic AMP (cAMP) were made during aerobic and anaerobic growth and upon an aerobic-to-anaerobic transition. Internal cAMP levels were similar (5 to 10 muM) throughout exponential growth, whether aerobic or anaerobic, but only during anaerobiosis was threonine dehydratase synthesized. When an exponentially growing aerobic culture was made anaerobic, a sharp increase in internal cAMP was noted, reaching 300 muM within 10 min and declining thereafter to normal anaerobic levels. Threonine dehydratase synthesis was detected immediately after the attainment of peak cAMP levels and continued for several generations. A similar pattern but with less accumulation of cAMP and less threonine dehydratase production was also noted upon treatment of an aerobically growing culture with KCN. Pyruvate addition at the time of anaerobic shock severely affected both cAMP accumulation and threonine dehydratase synthesis; however, externally added cAMP could partially counter the pyruvate effect on enzyme synthesis. The conclusion was reached that conditions which resulted in a temporary energy deficit brought about the major accumulation of cAMP, and this elevated level served as a signal for initiation of threonine dehydratase synthesis to supply energy by the nonoxidative degradation of threonine.
Aerobiosis↗
The role of glyoxylate in the regulation of biodegradative threonine dehydratase of Escherichia coli.
The activity of biodegradative threonine dehydratase of Escherichia coli K12 was reversibly inhibited by glyoxylate in the presence of AMP. Kinetic analysis showed that the inhibition was mixed with respect to L-threonine and competitive in terms of AMP; the inhibitory effect of glyoxylate was less pronounced at high protein concentrations. Incubation of dehydratase with L-threonine shifted the absorption maximum of the enzyme-bound pyridoxal phosphate from 413 to 425 nm; addition of glyoxylate completely prevented the threonine-mediated spectral shift. In addition to the inhibitory effect, incubation of purified enzyme with glyoxylate resulted in a progressive, irreversible inactivation of the enzyme and formation of inactive protein aggregates. The rates of inactivation were decreased with increasing concentrations of protein and AMP. During inactivation by glyoxylate, the 413-nm absorption maximum of the native enzyme was replaced by a new peak at 385 nm. Experiments with [14C]glyoxylate showed a rapid binding of 1 mol of glyoxylate per 147,000 g followed by a slow binding of 3 additional mol of glyoxylate; the glyoxylate-protein linkage was stable to acid precipitation and protein denaturants. Competition binding experiments revealed that pyruvate (which also inactivated the E. coli enzyme, Feldman, D.A., and Datta, P. (1975) Biochemistry 14, 1760-1767) did not interfere with the binding of glyoxylate or vice versa, suggesting that the two keto acids may occupy separate sites on the enzyme molecule. Nevertheless, experiments on enzyme inactivation using glyoxylate plus pyruvate reveal mutual interactions between these ligands in terms of lack of additive effect, retardation in the spectral shift due to glyoxylate, and stabilization of the enzyme in the presence and absence of AMP. We conclude from these results that the control of biodegradative threonine dehydratase is governed by a complex set of regulatory events resulting from reversible and irreversible association of these effectors with the enzyme molecule.
Adenosine Monophosphate↗
[Purification, molecular multiplicity and kinetic properties of "biosynthetic" L-threonine dehydratase from E. coli K-12].
"Biosynthetic" L-threonine dehydratase was purified to homogeneous state with yield 29% of total activity from E. coli K-12. The cells were disrupted by means of ultra sound. Nucleic acids and nucleoproteins were precipitated with protamine sulphate, the proteins were fractioned with (NH4)2SO4, by gel filtration through Sephadex G-25 followed by chromatography on DEAE-cellulose using stepways elution by changing the pH-values. The homogenity of the enzyme was shown by polyacrylamide gel disc electrophoresis in the presence of dodecylsulphate. The enzyme consists of equal subunits having a molecular weight about 57000. The polyacrylamide gel disc electrophoresis had shown that the native enzyme consists of a set of oligomeric forms. The multiplisity of molecular organization of the enzyme was relfected in complicated kinetic behavior: at pH greater than 9 on the plots of initial reaction rate (upsilon) versus initial substrate concentration ([S]0) there were four inflexion points (two intermediate plateaux) the position and deepness of which depended on enzyme concentration. Kinetic properties of the highly purified enzyme and the enzyme in crude cell extracts at pH 9.3 and 7.4 were identical. At pH 8,3 on the upsilon versus [S]0 plots appeared two inflexion points (one intermediate plateau), the position of which practically did not depend on enzyme concentration in the reaction mixture but strongly depended on the enzyme concentration in the stock solution. Repeated polyacrylamide gel disc electrophoresis of several oligomeric forms isolated by the first electrophoresis had shown that oligomeric forms underwent a slow polymerization. It is suggested that "biosynthetic" L-threonine dehydratase from E. coli K-12 is a set of multiple oligomeric forms having different kinetic parameters. Probably, each form of the enzyme has a "simple" kinetics characterized by hyperbolic or sigmoidal shape of upsilon versus [S]0 plots. The rate of equilibrium between the oligomeric forms is small in comparison with the enzyme reaction velosity, that lead to the complex kinetic curves appearing as a result of summing up the kinetics inherent to the individual forms.
Escherichia coli↗
Requirements for induction of the biodegradative threonine dehydratase in Escherichia coli.
Synthesis of the biodegradative L-threonine dehydratase in Escherichia coli, Crookes strain, was prevented by dissolved oxygen concentrations of 6 micrometer or greater. This effect was shown to be exerted solely on synthesis, rather than being the result of enzyme inactivation in vivo. In addition to an anaerobic environment, maximum enzyme synthesis was dependent upon the presence of a complete complement of amino acids, with omission of L-threonine, L-valine, or L-leucine producing the largest decreases in enzyme formation. L-Threonine, the most essential of the amino acid requirements, could be partially replaced by DL-allothreonine or alpha-ketobutyrate. Half-maximal stimulation of enzyme synthesis occurred with 0.4 mM threonine in the medium. The roles of anaerobiosis and amino acids are interpreted as being in accord with the concept that threonine dehydratase functions in anaerobic energy production under conditions of amino acid sufficiency.
Acetoacetates↗
Threonine metabolism in vivo: effect of threonine intake and prior induction of threonine dehydratase in rats.
The metabolic fate of threonine was investigated in young male rats fed 15% amino acid diets containing from 0.15% to 0.85% of L-threonine. Liver serine-threonine dehydratase (S-TDH) activity did not increase with increasing dietary threonine content. The level of threonine required for maximum weight gain was not greater than 0.55% of the diet (or about 600 mumoles/day). Tissue free threonine content of rats fed the diets with 0.15% or 0.3% of threonine was very low but increased sharply with increasing dietary threonine content above 0.3%. During ad libitum feeding of these diets containing L-[U-14C]threonine, rate of oxidation of threonine was low when intake was in the range of the requirement for maximum growth, but increased, thereafter as threonine intake increased. A 30-fold induction of liver S-TDH, by prior feeding of an 80% casein diet, did not result in increased oxidation of threonine when dietary threonine content was 0.15%. When dietary threonine content was increased to 0.5%, oxidation of threonine increased slightly but significantly. With 3% of threonine in the diet, rats previously fed a 15% casein diet had extremely high tissue threonine concentrations whereas those with high S-TDH activity, due to the previous feeding of the 80% casein diet, oxidized threonine rapidly and tissue threonine concentrations were elevated much less.
Animals↗
[Comparative kinetic properties of structure-bound and solubilized L-threonine dehydratase from brewer's yeast].
It has been shown that L-threonine dehydratase (EC 4.2.1.16) of brewer's yeast Saccharomyces carlsbergensis is localized in the mitochondrial fraction. The enzyme is easily solubilized from the mitochondria by changing the pH and ionic strength of the buffer. Some kinetic properties of structure-bound and solubilized L-threonine dehydratase have been compared at pH 6,5. The kinetic plots of the initial rate of the reaction versus initial substrate concentration for both enzymes have a hyperbolic shape; the affinities of both enzymes for the substrate appear to be similar (Km = 20 mM). Both enzymes are inhibited by L-isoleucine, the shape of the kinetic plots being thereby changed into sigmoidal. Solubilization results in a decrease of the mitochondral enzyme sensitivity to the inhibition by L-isoleucine and in an appearance of cooperative interactions between the allosteric sites.
Hydrogen-Ion Concentration↗
Catabolite inactivation of biodegradative threonine dehydratase of Escherichia coli.
Incubation of Escherichia coli cells with glucose, pyruvate, and certain other metabolites led to rapid inactivation of inducible biodegradative threonine dehydratase. Analysis with several mutant strains showed that pyruvate, and not a metabolite derived from pyruvate, was capable of inactivating enzyme, and that glucose acted indirectly after being converted to pyruvate. Some other alpha-keto acids such as oxaloacetate and alpha-ketobutyrate (but not alpha-ketoglutarate) were also effective. Inactivation of threonine dehydratase by pyruvate was also observed with purified enzyme preparations. The rates of enzyme inactivation increased with increased concentrations of pyruvate and decreased with increased levels of AMP. Increasing protein concentrations lowered the rates of enzyme inactivation. Dithiothreitol had a large effect on the maximum extent of inactivation of the enzyme by pyruvate; high concentrations of AMP and DTT almost completely counteracted the effect of pyruvate. Gel filtration data showed that pyruvate influenced the oligomeric state of the enzyme by altering the association-dissociation equilibrium in favor of dissociation; the Stokes' radius of the pyruvate-inactivated enzyme was 32 A as compared to 42 A for the untreated enzyme. Reassociation of the dissociated form of the enzyme was achieved by removal of excess free pyruvate by dialysis against buffer supplemented with AMP and DTT. Incubation of threonine dehydratase with [14-C]pyruvate revealed apparent covalent attachment of pyruvate to the enzyme. Strong protein denaturants such as guanidine, urea, and sodium dodecyl sulfate failed to release bound radioactive pyruvate; the molar ratio of firmly bound pyruvate was approximately 1 mol/150,000 g of protein. Pretreatment of the enzyme with p-chloromercuribenzoate and 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) did not reduce the binding of [14-C]pyruvate suggesting no active site SH was involved in the pyruvate-enzyme linkage. Titration of active and pyruvate-inactivated enzyme with Nbs2 indicated that the loss in enzyme activity was not due to oxidation of essential sulfhydryl groups on the enzyme. Based on these data we propose that the mechanism of enzyme inactivation by pyruvate involves covalent attachment of pyruvate to the active oligomeric form of the enzyme followed by dissociation of the oligomer to yield inactive enzyme.
Adenosine Monophosphate↗
Studies of homogeneous "biosynthetic" L-threonine dehydratase from Escherichia coli K-12. Some kinetic properties and molecular multiplicity.
"Biosynthetic" L-threonine dehydratase (EC 4.2.1.16) was purified to a homogeneous state with 29% yield of total activity from Escherichia coli K-12. The homogeneity of the enzyme was shown by polyacrylamide gel disc electrophoresis in the presence of dodecyl sulphate. The enzyme consisted of equal subunits having a molecular weight of about 57 000. The polyacrylamide gel disc electrophoresis has shown that the native enzyme consisted of a set of oligomeric forms. The multiplicity of molecular organization of the enzyme was reflected in complicated kinetic behaviour: at pH greater than 9 on the plots of initial reaction rate (v) versus initial substrate concentration ([S]o) there were four inflexion points (two intermediate plateaux), the position and deepness of which depended on enzyme concentration. At pH 8.3 on the v versus [S]o plots appeared two inflexion points (one intermediate plateu), the position of which practically did not depend on enzyme concentration in the reaction mixture, but strongly depended on the enzyme concentration in the stock solution. Repeated polyacrylamide gel disc electrophoresis of several oligomeric forms, isolated by the first electrophoresis, has shown that the oligomeric forms underwent a slow polymerization. It was suggested that "biosynthetic" L-threonine dehydratase from E. coli K-12 is a set of multiple oligomeric forms, having different kinetic parameters. Probably, each form of the enzyme has a "simple" kinetics characterized by hyperbolic or sigmoidal shape of v versus [S]o plots. The rate of equilibrium installation between the oligomeric forms was small in comparison with the enzyme reaction velocity, that lead to the complex kinetic curves, appearing as a result of summing up of the kinetics inherent to theindividual forms.
Escherichia coli↗
Inhibition of Escherichia coli biodegradative threonine dehydratase by pyruvate.
Pyruvate inhibits Escherichia coli K-12 biodegradative threonine dehydratase activity by a mechanism distinct from product inhibition by alpha-ketobutyrate and catabolite inactivation by intermediary metabolites.
Adenosine Monophosphate↗
Hepatic induction of threonine dehydratase and tryptophan pyrrolase to tyrotoxic rats.
The levels of hepatic threonine dehydratase and tryptophan pyrrolase are elevated in 5% tyrosine-fed rats, and these increases are dependent on the dietary tyrosine level. Experiments with RNA and protein synthesis inhibitors indicate that the appearance of these new enzyme activities are dependent on concomitant new protein synthesis and the inducer operates at a transcriptional level.
Animals↗
Biodegradative threonine dehydratase. Reduction of ferricyanide by an intermediate of the enzyme-catalyzed reaction.
The threonine-dependent reduction of ferricyanide catalyzed by the purified biodegradative threonine dehydratase of Escherichia coli has been studied. The rate of production of 2-oxobutyrate in the presence of ferricyanide was lower than that found in the absence of ferricyanide. The concentrations of threonine required for half-maximal effects for the reduction of ferricyanide and, in the presence of the dye, for 2-oxobutyrate production, were 3 mM and 9mM, respectively. Reduction of ferricyanide was accompanied by evolution of CO2, and even within a very short incubation time with the enzyme, the ratio of ferricyanide reduced over CO2 evolved was approximately 7. Stopping the enzyme activity after a brief exposure to threonine at pH 9.7 resulted in the accumulation of an intermediate (with a half-life of 4 min at 25 degrees C) which formed an adduct with N-ethylmaleimide; the accumulated intermediate, in the absence of N-ethylmaleimide, reduced ferricyanide with concomitant evolution of CO2. We conclude from these results that 2-aminocrotonate is the intermediate which serves as a source of reducing equivalent for ferricyanide, and nonstoichiometric amount of ferricyanide reduction may be attributed to some secondary reactions of ferricyanide with compounds derived from the oxidation product of 2-aminocrotonate.
Escherichia coli↗
[Intermediate plateaux in kinetics of the reaction catalyzed by biodegradative L-threonine dehydratase from Escherichia coli].
It has been shown that for the reaction catalyzed by "biodegradative" L-threonine dehydratase from E. coli strains K-12 and 980 in 0.5 M phosphate-carbonate buffer, pH 8.4 and pH 9.5, the plots of initial reaction rate (v) versus the initial substrate concentration ([S]0 are characterized by several inflection points, i. e. an intermediate plateau. The plot of v versus the allosteric activator (AMP) concentration have very complicated shapes: there are several inflection points, and also the maximum at L-threonine concentration equal to 3-10(2) and 5-10(-2) M. High AMP concentrations inhibit the enzyme at high substrate concentrations. The reduced glutathion dose not influence the enzyme and does not alter the activating effect of AMP. On the basis of the data obtained it is proposed that the substrate and AMP shift the equilibrium between multiple oligomeric enzyme forms differing in catalytic activity and kinetic manifestations of allosteric interactions between the active and allosteric AMP-binding sites towards polymerization. Thus, the functioning the enzyme under study is discussed in the frames of the model of dissociating regulatory enzymes with multiple intermediate oligomeric forms.
Adenosine Monophosphate↗
[Effect of higher alcohols on the activity of biosynthetic L-threonine dehydratase from Saccharomyces carlsbergensis brewer's yeast].
The effects of low (1 . 10(-4) M) and high (1 . 10(-3) M) concentrations of n-propanol, isobutanol and isoamylols on the kinetic behaviour of "biosynthetic" L-threonine dehydratase from brewer's yeast S. carlsbergensis 776 were studied. It was concluded that these alcohols control the activity of the first enzyme of the L-threonine biosynthetic pathway.
Alcohols↗
[The serine- and threonine dehydratases of brucellae].
In studying 62 strains of brucellae of all the known species an inductive enzyme--1-serine-hydrolyase(desaminating) was revealed in the intact cells and cell-free extracts. The activity of this enzyme depended on the species of brucellae and showed a sharp elevation in destruction of the cells with supersonic waves. Intact brucellae failed to desaminate treonine. A constitution enzyme 1-treonine-hydrolase (desaminating) was found in the cell-free extracts of Br. neotomae and brucellae isolated from murine rodents of the Northern Caucasus. Some properties of both enzymes were studied.
Ammonia↗
Spinach threonine dehydratase. Inhibition by L-cysteine and D-cysteine.
L-threonine deaminase from spinach is inhibited by D- and L-cysteine. The inhibition patterns by D-cysteine and by L-cysteine are non-competitive. The value of Ki for D-cysteine and L-cysteine is of the same order of magnitude. Inhibitions by L-isoleucine and L-cysteine are additional. These results indicate that inhibition by L-cysteine occurs on a site of enzyme which is different from the binding site for L-isoleucine and L-valine. Probably L-cysteine and D-cysteine form the thiazolidinic ring with a PLP mole. Which is not costituent of the active site, but located in a different region.
Binding Sites↗
Allosteric inhibition and catabolite inactivation of purified biodegradative threonine dehydratase of Salmonella typhimurium.
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Adenosine Monophosphate↗
Correlation between generation times and L-threonine dehydratase activities in isoleucine revertants of Serratia marcescens.
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Acetolactate Synthase↗