Regulation of Pyrimidine Biosynthesis in Saccharomyces cerevisiae

Biochemical steps of the pyrimidine pathway have been found to be the same in yeast as in bacteria, and all except one step have been characterized. The activities of the first two enzymes, carbamoyl phosphate synthetase and aspartic transcarbamylase, are simultaneously controlled by feedback inhibition and repression. Moreover, these enzymes are coded by the same genetic region (ura-2) and seem to form a single enzymatic complex. The enzymes that follow later in the pathway are induced in a sequential way by the intermediary products and are insensitive to pyrimidine repression. The corresponding genes (ura-4, ura-1, ura-3) are not linked to each other or to ura-2, the gene for carbamoyl phosphate synthetase and aspartic transcarbamylase. Mutants that have simultaneously lost feedback inhibition by uridine triphosphate for carbamoyl phosphate synthetase and for aspartic transcarbamylase have been found and mapped in the gene ura-2.     

Enzymatic synthesis of polymethylated flavonols in Chrysosplenium americanum. II. Substrate interaction and product inhibition studies of flavonol 3-, 6-, and 4'-O-methyltransferases

The steady-state kinetic behavior of three position-specific O-methyltransferases (3-, 4'-, and 6-OMTs) was compared with reference to substrate inhibition patterns in Chrysosplenium americanum. The 6-OMT was severely inhibited by the flavonoid substrate at concentrations close to Km, whereas the other two enzymes were less affected by their respective flavonoid substrates. Substrate interaction kinetics for the 6-OMT gave converging lines consistent with a sequential binding mechanism, whereas the data generated for the 3- and 4'-OMTs could be fitted to the equation for a ping-pong mechanism or to that of a sequential binding mechanism where Kia was much smaller Ka. More information on the mechanism of reaction was obtained from product inhibition studies. The three enzymes exhibited competitive inhibition patterns between S-adenosyl-L-methionine (SAM) and S-adenosyl-L-homocysteine (SAH), whereas other patterns were either noncompetitive or uncompetitive. The steady-state kinetic properties of the 3-, 4'-, and 6-OMTs were consistent with a sequential ordered reaction mechanism, in which SAM and SAH were leading reaction partners and included an abortive EQB complex. Product inhibition constants were sufficiently low to suggest that these may be important in regulating the pathway of polymethylated flavonoid synthesis. It was suggested that due to their greater sensitivity to inhibition by SAH, the OMTs involved in earlier steps of the methylation sequence may regulate the rate of synthesis of final products in Chrysosplenium.     

Keeping the receptor tyrosine kinase signaling pathway in check: lessons from Drosophila

The receptor tyrosine kinase (RTK) signaling network plays a central role in regulating cellular differentiation, proliferation, and survival in all metazoan animals. Excessive or continuous activation of the RTK pathway has been linked to carcinogenesis in mammals, underscoring the importance of preventing uncontrolled signaling. This review will focus on the inhibitory mechanisms that keep RTK-mediated signals in check, with emphasis on conserved principles discerned from studies using Drosophila as a model system. Two general strategies of inhibition will be discussed. The first, threshold regulation, postulates that an effective way of antagonizing RTK signaling is to erect and maintain high threshold barriers that prevent inappropriate responses to moderate signaling levels. Activation of the pathway above this level overcomes the inhibitory blocks and shifts the balance to allow a positive flow of inductive information. A second layer of negative regulation involving induction of negative feedback loops that limit the extent, strength, or duration of the signal prevents runaway signaling in response to the high levels of activation required to surmount the threshold barriers. Such autoinhibitory mechanisms attenuate signaling at critical points throughout the network, from the receptor to the downstream effectors.     

Control of the flux in the arginine pathway of Neurospora crassa. Modulations of enzyme activity and concentration.

The influence of particular enzyme activities on the flux of metabolites in a pathway can be estimated by 'modulating' enzymes (i.e. changing turnover or concentration) and measuring the response in various parts of the system. By controlling the nuclear ration of two genetically different nuclear types in heterokaryons, the enzyme concentrations at four different steps in the arginine pathway were decreased over a range. This range was extended by the use of bradytrophs, mutant strains specifying enzymes with greatly diminished enzyme activities. Strains altered simultaneously at more than one step were also constructed by genetic recombination. By measuring the outputs of the pathway and the steady-state concentrations of intermediate pools, the fluxes in different parts of the pathway were calculated. This allowed the construction of flux/enzyme relationships, the slope of which is a measure of the sensitivity of a flux to the change in enzyme activity at that step. All fluxes were found to be considerably buffered for quite substantial decreases in the activities of all enzymes. Mass action plays an important part in this phenomenon, as do inhibition and repression. Because of the existence of expansion fluxes in growing systems, we find quantitatively different fluxes in different parts of the single pathway. For the same reason some enzyme modulations given decreased fluxes in one part and increased fluxes in another. The understanding of control in the pathway thus involves consideration of many mechanisms operating simultaneously and the estimation of changes in the whole system. The concept of a 'rate-limiting step' is found to be inadequate and is replaced by a quantitative measure, the Sensitivity Coefficient, which takes account of all the interactions. It is shown that control of the flux is shared among all the enzymes of the pathway. The results are discussed in terms of the theory of flux control.     

A mathematical modelling framework for understanding chemorepulsive signal transduction in Dictyostelium

Chemorepulsion is the process by which an organism or a cell moves in the direction of decreasing chemical concentration. While a few experimental studies have been performed, no mathematical models exist for this process. In this paper we have modelled gradient sensing, the first subprocess of chemorepulsion, in Dictyostelium discoideum-a well characterized model eukaryotic system. We take the first steps towards achieving a comprehensive mechanistic understanding of chemorepulsion in this system. We have used, as a basis, the biochemical network of the Keizer-Gunnink et al. (2007) to develop the mathematical modelling framework. This network describes the underlying pathways of chemorepellent gradient sensing in D. discoideum. Working within this modelling framework we address whether the postulated interactions of the pathways and species in this network can lead to a chemorepulsive response. We also analyse the possible role of additional regulatory effects (such as additional receptor regulation of enzymes in this network) and if this is necessary to achieve this behaviour. Thus we have investigated the receptor regulation of important enzymes and feedback effects in the network. This modelling framework generates important insights into and testable predictions regarding the role of key components and feedback loops in regulating chemorepulsive gradient sensing, and what factors might be important for generating a chemorepulsive response; it serves as a first step towards a comprehensive mechanistic understanding of this process.     

Effect of mutation in the aromatic amino acid pathway on sporulation of Saccharomyces cerevisiae.

Mutations in ARO1 and ARO2 genes coding for enzymes involved in the common part of the aromatic amino acid pathway completely block the sporulation of Saccharomyces cerevisiae when in a homozygous state, whereas mutations in all the other genes of the same pathway do not. This effect is not due to the lack of any intermediate metabolite but rather to the accumulation of a metabolite preceding chorismic acid. Shikimic acid or one of its precursors was identified as the possible inhibitor. The presence of the three aromatic amino acids in the sporulation medium restores the ability to undergo meiosis. This seems not to be due to a feedback inhibition of the first enzymes of the pathway but rather to a competition between aromatic amino acids and the inhibitor on a site specific for the meiotic process. The inhibition of sporulation seems to occur at a very early step in meiosis, as indicated by the lack of premeiotic DNA synthesis in aro1 and aro2 mutants.     

Information Transfer in Gonadotropin-releasing Hormone (GnRH) Signaling: EXTRACELLULAR SIGNAL-REGULATED KINASE (ERK)-MEDIATED FEEDBACK LOOPS CONTROL HORMONE SENSING*

Cell signaling pathways are noisy communication channels, and statistical measures derived from information theory can be used to quantify the information they transfer. Here we use single cell signaling measures to calculate mutual information as a measure of information transfer via gonadotropin-releasing hormone (GnRH) receptors (GnRHR) to extracellular signal-regulated kinase (ERK) or nuclear factor of activated T-cells (NFAT). This revealed mutual information values <1 bit, implying that individual GnRH-responsive cells cannot unambiguously differentiate even two equally probable input concentrations. Addressing possible mechanisms for mitigation of information loss, we focused on the ERK pathway and developed a stochastic activation model incorporating negative feedback and constitutive activity. Model simulations revealed interplay between fast (min) and slow (min-h) negative feedback loops with maximal information transfer at intermediate feedback levels. Consistent with this, experiments revealed that reducing negative feedback (by expressing catalytically inactive ERK2) and increasing negative feedback (by Egr1-driven expression of dual-specificity phosphatase 5 (DUSP5)) both reduced information transfer from GnRHR to ERK. It was also reduced by blocking protein synthesis (to prevent GnRH from increasing DUSP expression) but did not differ for different GnRHRs that do or do not undergo rapid homologous desensitization. Thus, the first statistical measures of information transfer via these receptors reveals that individual cells are unreliable sensors of GnRH concentration and that this reliability is maximal at intermediate levels of ERK-mediated negative feedback but is not influenced by receptor desensitization.     

Periodic metabolic systems: Oscillations in multiple-loop negative feedback biochemical control networks

Summary For a general multiple loop feedback inhibition system in which the end product can inhibit any or all of the intermediate reactions it is shown that biologically significant behaviour is always confined to a bounded region of reaction space containing a unique equilibrium. By explicit construction of a Liapunov function for the general n dimensional differential equation it is shown that some values of reaction parameters cause the concentration vector to approach the equilibrium asymptotically for all physically realizable initial conditions. As the parameter values change, periodic solutions can appear within the bounded region. Some information about these periodic solutions can be obtained from the Hopf bifurcation theorem. Alternatively, if specific parameter values are known a numerical method can be used to find periodic solutions and determine their stability by locating a zero of the displacement map. The single loop Goodwin oscillator is analysed in detail. The methods are then used to treat an oscillator with two feedback loops and it is found that oscillations are possible even if both Hill coefficients are equal to one.     

Tryptophan biosynthesis in Saccharomyces cerevisiae: control of the flux through the pathway.

Enzyme derepression and feedback inhibition of the first enzyme are the regulatory mechanisms demonstrated for the tryptophan pathway in Saccharomyces cerevisiae. The relative contributions of the two mechanisms to the control of the flux through the pathway in vivo were analyzed by (i) measuring feedback inhibition of anthranilate synthase in vivo, (ii) determining the effect of regulatory mutations on the level of the tryptophan pool and the flux through the pathway, and (iii) varying the gene dose of individual enzymes of the pathway at the tetraploid level. We conclude that the flux through the pathway is adjusted to the rate of protein synthesis by means of feedback inhibition of the first enzyme by the end product, tryptophan. The synthesis of the tryptophan enzymes could not be repressed below a basal level by tryptophan supplementation of the media. The enzymes are present in excess. Increasing or lowering the concentration of individual enzymes had no noticeable influencing on the overall flux to tryptophan. The uninhibited capacity of the pathway could be observed both upon relieving feedback inhibition by tryptophan limitation and in feedback-insensitive mutants. It exceeded the rate of consumption of the amino acid on minimal medium by a factor of three. Tryptophan limitation caused derepression of four of the five tryptophan enzymes and, as a consequence, led to a further increase in the capacity of the pathway. However, because of the large reserve capacity of the "repressed" pathway, tryptophan limitation could not be imposed on wild-type cells without resorting to the use of analogs. Our results, therefore, suggest that derepression does not serve as an instrument for the specific regulation of the flux through the tryptophan pathway.     

Irreversibility in unbranched pathways: preferred positions based on regulatory considerations

It has been observed experimentally that most unbranched biosynthetic pathways have irreversible reactions near their beginning, many times at the first step. If there were no functional reasons for this fact, then one would expect irreversible reactions to be equally distributed among all positions in such pathways. Since this is not the case, we have attempted to identify functional consequences of having an irreversible reaction early in the pathway. We systematically varied the position of the irreversible reaction in model pathways and compared the resulting systemic behavior according to several criteria for functional effectiveness, using the method of mathematically controlled comparisons. This technique minimizes extraneous differences in systemic behavior and identifies those that are fundamental. Our results show that a pathway with an irreversible reaction located at the first step, and with all other reactions reversible, is on average better than an otherwise equivalent pathway with all reactions reversible, which in turn is on average better than an otherwise equivalent pathway with an irreversible reaction located at any step other than the first. Pathways with an irreversible first reaction and low concentrations of intermediates (one of the primary criteria for functional effectiveness) exhibit the following profile when compared to fully reversible pathways: changes in the concentration of intermediates in response to changes in the level of initial substrate are equally low, the robustness of the intermediate concentrations and of the flux is similar, the margins of stability are similar, flux is more responsive to changes in demand for end product, intermediate concentrations are less responsive to changes in demand for end product, and transient times are shorter. These results provide a functional rationale for the positioning of irreversible reactions at the beginning of unbranched biosynthetic pathways.     

Lysine accumulation in maize cell cultures transformed with a lysine-insensitive form of maize dihydrodipicolinate synthase

Lysine is one of the nutritionally limiting amino acids in food and feed products made from maize (Zea mays L.). Two enzymes in the lysine biosynthesis pathway, aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS), have primary roles in regulating the level of lysine accumulation in plant cells because both enzymes are feedback-inhibited by lysine. An isolated cDNA clone for maize DHPS was modified to encode a DHPS much less sensitive to lysine inhibition. The altered DHPS cDNA was transformed into maize cell suspension cultures to determine the effect on DHPS activity and lysine accumulation. Partially purified DHPS (wildtype plus mutant) from transformed cultures was less sensitive to lysine inhibition than wild-type DHPS from nontransformed cultures. Transformed cultures had cellular free lysine levels as much as four times higher than those of nontransformed controls. Thus, we have shown that reducing the feedback inhibition of DHPS by lysine can lead to increased lysine accumulation in maize cells. Increasing the capacity for lysine synthesis may be an important step in improving the nutritional quality of food and feed products made from maize.     

Increasing the flux in metabolic pathways: A metabolic control analysis perspective

The problems of engineering increased flux in metabolic pathways are analyzed in terms of the understanding provided by metabolic control analysis. Over-expression of a single enzyme is unlikely to be effective unless it is known to have a high flux control coefficient, which can be used as an approximate predictive tool. This is likely to rule out enzymes subject to feedback inhibition, because it transfers control downstream from the inhibited enzyme to the enzymes utilizing the feedback metabolite. Although abolishing feedback inhibition can restore flux control to an enzyme, it is also likely to cause large increases in the concentrations of metabolic intermediates. Simultaneous and coordinated over-expression of most of the enzymes in a pathway can, in principle, produce substantial flux increases without changes in metabolite levels, though technically it may be difficult to achieve. It is, however, closer to the method used by cells to change flux levels, where coordinated changes in the level of activity of pathway enzymes are the norm. Another option is to increase the demand for the pathway product, perhaps by increasing its rate of excretion or removal. Copyright 1998 John Wiley & Sons, Inc.     

Regulation of uridylic acid biosynthesis in the cyanobacterium Anabaena variabilis.

The pathway of uridylic acid biosynthesis established by Leiberman, Kornberg, and Simms has been shown to be operative in the filamentous cyanobacterium Anabaena variabilis. The only enzyme of uridylic acid biosynthesis found to be lacking in two uracil-requiring strains of A. variabilis was aspartate transcarbamylase, the first enzyme in the pathway of de novo biosynthesis of uridvlic acid. Neither uracil-limited growth of a uracil-requiring mutant nor growth of the wild type in high concentrations of uracil resulted in substantial changes in the specific activities of enzymes of uridylic acid biosynthesis. It is therefore concluded that A. variabilis does not regulate all enzymes of this pathway by means of repression. However, control of the flow of intermediates through this pathway is possible by feedback inhibition of aspartate transcarbamylase by a variety of nucleotides.     

Regulatory mechanism of histidine-tagged homocitrate synthase from Saccharomyces cerevisiae. I. Kinetic studies

Homocitrate synthase (HCS) catalyzes one of the regulated steps of the alpha-aminoadipate pathway for lysine biosynthesis in fungi. The kinetic mechanism of regulation of HCS from Saccharomyces cerevisiae by Na+ and the feedback inhibitor lysine was studied by measuring the initial rate in the absence and presence of the effectors. The data suggest that Na+ is an activator at low concentrations and an inhibitor at high concentrations and that these effects occur as a result of the monovalent ion binding to two different sites in the free enzyme. Inhibition and activation by Na+ can occur simultaneously, with the net rate of the enzyme determined by Na+/K(iNa+) and Na+/K(act), where K(iNa+) and K(act) are the inhibition and activation constants, respectively. The inhibition by Na+ was eliminated at high concentrations of acetyl-CoA, the second substrate bound, but the activation remained. Fluorescence binding studies indicated that lysine bound with high affinity to its binding site as an inhibitor. The inhibition by lysine was competitive versus alpha-ketoglutarate and linear in the physiological range of lysine concentrations up to 5 mm. The effects of Na+ and lysine were independent of one another. A model is developed for regulation of HCS that takes into account all of the effects discussed above.     

The serine acetyltransferase reaction: acetyl transfer from an acylpantothenyl donor to an alcohol

Serine acetyltransferase is a member of the left-handed parallel beta-helix family of enzymes that catalyzes the committed step in the de novo synthesis of l-cysteine in bacteria and plants. The enzyme has an ordered kinetic mechanism with acetyl CoA bound prior to l-serine and O-acetyl-l-serine released prior to CoA. The rate-limiting step along the reaction pathway is the nucleophilic attack of the serine hydroxyl on the thioester of acetyl CoA. Product release contributes to rate-limitation at saturating concentrations of reactants. The reaction is catalyzed by an active site general base with a pK of 7, which accepts a proton from the serine hydroxyl as a tetrahedral intermediate is formed between the reactants, and donates it to the thiol of CoA as the intermediate collapses to give products. This mechanism is likely the same for all O-acyltransferases that catalyze their reaction by direct attack of the alcohol on the acyl donor, using an active-site histidine as the general base. Serine acetyltransferase is regulated by feedback inhibition by the end product l-cysteine, which acts by binding to the serine site in the active site and inducing a conformational change that prevents reactant binding. The enzyme also associates with O-acetylserine sulfhydrylase, the final enzyme in the biosynthetic pathway, which contributes to stabilizing the acetyltransferase.     

Alkyl selenosulphates (seleno Bunte salts). A new class of thiol-blocking reagents.

Potassium benzyl selenosulphate and potassium p-nitrobenzyl selenosulphate were shown to be powerful inhibitors of the thiol-dependent enzymes glutathione reductase and papain, but to have no effect on the serine-dependent proteinase trypsin. By contrast, potassium benzyl thiosulphate and potassium p-nitrobenzyl thiosulphate, at much higher concentrations, have virtually no effect on any of the enzymes. The selenosulphates show characteristics of both reversible non-competitive and irreversible inhibition. On the basis of model reactions in which the selenosulphates react instantly with cysteine, it is suggested that they form labile selenosulphide derivatives with the enzymes, but that these derivatives may be broken down either by the normal functioning of the enzyme (in the case of glutathione reductase) or by the approaching substrate (in the case of papain). Continued inhibition of the enzymes requires a stoicheiometric excess of inhibitor over enzyme.     

Comparative action of glyphosate as a trigger of energy drain in eubacteria.

Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa, each possessing a 5-enolpyruvylshikimate 3-phosphate synthase that is sensitive to inhibition by glyphosate [N-(phosphonomethyl)glycine], provide a good cross-section of organisms exemplifying the biochemical diversity of the aromatic pathway targeted by this potent antimicrobial compound. The pattern of growth inhibition, the alteration in levels of aromatic-pathway enzymes, and the accumulation of early-pathway metabolites after the addition of glyphosate were distinctive for each organism. Substantial intracellular shikimate-3-phosphate accumulated in response to glyphosate treatment in all three organisms. Both E. coli and P. aeruginosa, but not B. subtilis, accumulated near-millimolar levels of shikimate-3-phosphate in the culture medium. Intracellular backup of common-pathway precursors of shikimate-3-phosphate was substantial in B. subtilis, moderate in P. aeruginosa, and not detectable in E. coli. The full complement of aromatic amino acids prevented growth inhibition and metabolite accumulation in E. coli and P. aeruginosa where amino acid end products directly control early-pathway enzyme activity. In contrast, the initial prevention of growth inhibition in the presence of aromatic amino acids in B. subtilis was succeeded by progressively greater growth inhibition that correlated with rapid metabolite accumulation. In B. subtilis glyphosate can decrease prephenate concentrations sufficiently to uncouple the sequentially acting loops of feedback inhibition that ordinarily link end product excess to feedback inhibition of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase by prephenate. The consequential unrestrained entry is an energy-rich substrates into the aromatic pathway, even in the presence of aromatic amino acid end products, is an energy drain that potentially accounts for the inability of end products to fully reverse glyphosate inhibition in B. subtilis. Even in E. coli after glyphosate inhibition and metabolite accumulation were allowed to become fully established, a transient period where end products were capable of only partial reversal of growth inhibition occurred. The distinctive metabolism produced by dissimilation of different carbon sources also profound effects upon glyphosate sensitivity.     

Control of aromatic acid biosynthesis in Bacillus subtilis: sequenial feedback inhibition

Nester, E. W. (University of Washington, Seattle), and R. A. Jensen. Control of aromatic acid biosynthesis in Bacillus subtilis: sequential feedback inhibition. J. Bacteriol. 91:1594-1598. 1966.-The three major end products of aromatic acid synthesis, tyrosine, phenylalanine, and tryptophan, were tested for their ability to inhibit the first enzymes of the three terminal branches of the pathway as well as the enzyme common to both tyrosine and phenylalanine synthesis. Tyrosine inhibits the activity of prephenate dehydrogenase and also prephenate dehydratase to a limited extent. Phenylalanine inhibits the activity of prephenate dehydratase and, at much higher concentrations, prephenate dehydrogenase. Tryptophan inhibits the activity of anthranilate synthetase and, to some extent, prephenate dehydrogenase and prephenate dehydratase. Chorismate mutase is not inhibited by either 1 mm tyrosine or 1 mm phenylalanine when these are present singly or together in the reaction mixture. The significance of the feedback control of the terminal branches to the feedback control of that part of the pathway common to the synthesis of all three amino acids is discussed.     

Marked differences in the swainsonine inhibition of rat liver lysosomal alpha-D-mannosidase, rat liver Golgi mannosidase II, and jack bean alpha-D-mannosidase

Swainsonine, a plant toxin, strongly inhibits certain alpha-D-mannosidases but has no effect on others [D. R. P. Tulsiani, T. M. Harris, and O. Touster (1982) J. Biol. Chem. 257, 7936-7939]. The reversible inhibition of jack bean and lysosomal alpha-D-mannosidases has previously been suggested to be similar in nature but quite complex. Specific differences in the action of swainsonine on these two enzymes and on Golgi mannosidase II are reported. (a) The inhibition of the jack bean mannosidase, but not rat liver lysosomal alpha-D-mannosidase or Golgi mannosidase II, is increased by preincubation with the alkaloid. (b) The inhibition of the jack bean and lysosomal enzymes, but not mannosidase II, is competitive at inhibitor concentrations of less than or equal to 0.5 microM. (c) The inhibition of jack bean alpha-mannosidase is largely irreversible, its very limited reversibility being partially dependent upon the swainsonine concentration used and on the time of preincubation with the inhibitor. On the other hand, the inhibition of lysosomal alpha-mannosidase is largely reversible, as shown by dilution experiments and by the use of [3H]swainsonine. Golgi mannosidase II shows intermediate reversibility, the results indicating two modes of binding; one rapid and irreversible, the other much slower and reversible.     

Phenolic metabolism in petunia tissues. I. Characteristic responses of enzymes involved in different steps of polyphenol synthesis to different hormonal influences.

Pronounced changes in enzymatic patterns occur in petunia tissues when calluses are subcultured on media containing different growth substances. As judged by variations of enzymes related to primary metabolism (6-phosphogluconate and malate dehydrogenases) there are individual responses for each metabolic pathway. Concerning the enzymes of aromatic metabolism: (a) Phenylalanine ammonia-lyase, cinnamate and p-coumarate hydroxylases and the enzyme(s) activating phenylpropanoid units vary in the same manner. (b) Chalcone-flavanone isomerase, a key enzyme in the synthesis of flavonoids, and coniferyl alcohol dehydrogenase, which leads to the monomers of lignins, have, on the other hand, an independent behaviour. These responses show that the enzymes involved in the synthesis and activation of phenylpropanoid units seem to act coordinately in plants. Moreover, the data suggest that the common pathway leading to the activated cinnamic acids and the specific metabolic steps of lignin and flavonoid synthesis are regulated in a different way.