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PubMedDate not supplied

Allosteric regulation of beef liver arginase activity by L-ornithine.

Inhibition of beef liver arginase by L-ornithine was investigated with two sets of independent experiments. Progress curves of the production of urea were simulated with two integrated Michaelis-Menten equations for competitive and non-competitive inhibition by ornithine. Both fitted the curves well, but failed to correctly predict the inhibition when the reaction was started with ornithine already present. Measurement of initial rates of reaction enabled an allosteric model to be built in accordance to Monod-Wyman-Changeux: arginine preferentially binds to the active state R and ornithine preferentially binds to the inactive state T. In the absence of both ligands, the R in equilibrium T equilibrium slightly favours the active state and both states bind ornithine more strongly than arginine. No great variation was observed in the 6 parameters of the model by assuming the enzyme to be a trimer or a tetramer. The model was able to predict not only the initial rate curves, from which it was derived, but also the progress curves independently obtained.

Allosteric Regulation↗
PubMed1978

[Allosteric regulation of ceruloplasmin activity].

Study of the interactions of homogenous human ceruloplasmin preparations with histamine show that the rate of p-phenylene diamine oxidation by ceruloplasmin is increased in the presence of histamine; the increase in the enzyme activity is independent of histamine concentration. The dependence of the reaction rate on substrate concentration is S-shaped, both in the presence and in the absence of histamine. The respective values of the Hill coefficient and Rs for the enzyme in the presence and in the absence of histamine are 2.5 and 2.0 and 8.0 and 10.4. Histamine does not change ceruloplasmin-specific absorption at 610 nm. Evidence from EPR studies show that histamine does not interact with Cu of the enzyme active center. During interaction with histamine the antigenic properties of the enzyme are changed. Histamine increases the oxidase activity of the enzyme in human and rat blood sera and exerts multifold effects on the enzyme activity in patients with hepatolenticular degeneration. After injection of histamine to rats the enzyme activity is increased without a simultaneous increase in Cu concentration in the blood serum, i.e. without de novo synthesis of ceruloplasmin. The data obtained suggest that ceruloplasmin is probably an allosteric enzyme, which histamine is its positive allosteric effector.

Allosteric Regulation↗
PubMed1978

Subunit dissociation in the allosteric regulation of glycerol kinase from Escherichia coli. 2. Physical evidence.

The dependence of the molecular weight of glycerol kinase on enzyme concentration and on binding of fructose 1,6-bisphosphate has been examined by velocity sedimentation, gel filtration, and polyacrylamide gel electrophoresis. The sedimentation coefficient and Stokes radius decrease as a consequence of dilution in a manner consistent with dissociation into half-molecules, with limiting values suggesting molecular weights of about 218,000 and 136,000 for the associated and dissociated species, respectively. Fructose 1,6-bisphosphate (5 mM) prevents the decrease in sedimentation coefficient brought about by dilution, suggesting a decrease in the apparent subunit dissociation constant of at least four orders of magnitude. Electrophoretic mobility in polyacrylamide gels increases as a consequence of dilution in the absence, but not in the presence, of fructose 1,6-bisphosphate. Ferguson plots indicate that glycerol kinase has the same molecular weight in the presence of fructose 1,6-bisphosphate as the covalently cross-linked tetramer and is substantially smaller in the absence of fructose 1,6-bisphosphate. These results are consistent with the model of glycerol kinase proposed in the preceding paper of this issue [de Riel, J.K., and Paulus, H. (1978), Biochemistry 17] relating subunit dissociation and ligand binding.

Allosteric Regulation↗
PubMed1978

Allosteric regulation of monocyclic interconvertible enzyme cascade systems: use of Escherichia coli glutamine synthetase as an experimental model.

The interconversion of Escherichia coli glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] between its adenylylated and unadenylylated forms has been used to verify the prediction derived from a theoretical analysis of the steady-state functions of a model for a monocyclic interconvertible enzyme cascade system [Stadtman, E. R. & Chock, P. B. (1977) Proc. Natl. Acad. Sci. USA 74, 2761-2770]. Because glutamine and alpha-ketoglutarate are multifunctional effectors and because three active enzyme complexes are involved in both adenylylation and deadenylylation of glutamine synthetase, at least 28 constants are required to describe the glutamine synthetase monocyclic cascade. Of these, 22 constants were determined experimentally and 6 were estimated via computer curve fitting. Despite the complexity, when both adenylylation and deadenylylation reactions are functioning, the number of adenylyl groups bound per mole of enzyme, n, assumes a steady-state level as is predicted by the model. This n value is determined by the mole fraction of P(IIA)-given by ([P(IIA)]/([P(IIA)] + [P(IID)])-and the ratio of glutamine to alpha-ketoglutarate (P(IID) and P(IID) are the unmodified and the uridylylated forms of the P(II) regulatory protein). In the presence of 0.5 mM glutamine and 2 mM alpha-ketoglutarate, the value of n increases as a nearly hyperbolic function in response to increasing mole fractions of P(IIA). When the constant level of alpha-ketoglutarate is gradually increased to 40 muM, the hyperbolic function converts slowly to a parabolic function. When the P(IIA) mole fraction was maintained at 0.6 and alpha-ketoglutarate levels were varied from 1 mM to 4 muM, an 800-fold increase in signal amplification was observed with respect to glutamine activation. In addition, because glutamine activates the adenylylation and inhibits the deadenylylation reaction, a sensitivity index of 2.1 (corresponding to a Hill number of 1.5) was obtained for the variation of n values in response to increasing glutamine concentration.

Adenosine Monophosphate↗
PubMed1975

Non-allosteric regulation of the uridine kinase from seeds of Zea mays.

Uridine kinase (ATP: uridine 5'-phosphotransferase, EC 2.7.1.48) has been partially purified from ungerminated hybrid corn seed. It is associated with a soluble high molecular weight fraction from which it apparently cannot be dissociated without loss of activity. The stability of the enzyme is enhanced by the addition of dithiothreitol, glycerol and nucleotide substrate. The nucleoside specificity of the enzyme is limited to nucleosides containing pyrimidine and ribose moieties, such as uridine and cytidine. High concentrations of nucleosides cause substrate inhibition, however. The Km values for uridine and cytidine are 53 muM and 125 muM, respectively, and under subsaturating conditions uridine is phosphorylated about five times faster than cytidine. The reaction follows an ordered Bi Bi kinetic pattern, with ATP and ADP in competition for the free form of the enzyme. Purine, but not pyrimidine, nucleoside triphosphates serve as phosphate donors without regard to the sugar moiety. However, all of these triphosphates appear to compete for the same site on the enzyme. (Km ATP equals 590 muM, Km (app) GTP equals 61 muM, and CTP and UTP are linear competitive inhibitors against ATP, with Ki values of 60 muM and 240 muM, respectively.) Therefore, end product control of uridine kinase apparently does not involve allosteric sites, but instead is envisioned as simple competition between relatively effective or ineffective phosphate donors for a position on the enzyme.

Kinetics↗
PubMed1977

Biosynthesis of bacterial glycogen: genetic and allosteric regulation of glycogen biosynthesis in Salmonella typhimurium LT-2.

Structural gene mutants of the glycogen biosynthetic enzymes adenosine diphosphate glucose pyrophosphorylase (glgC) and glycogen synthase (glgA) were isolated and partially characterized. The cotransduction frequencies of these genes with the aspartic semialdehyde dehydrogenase (asd) and glycerol-3-phosphate dehydrogenase (glpD) genes suggested the unambiguous gene order of glpD glgA glgC asd. The results of the three-factor cross glpD- glgA- glgC+ X glpD+ glgA+ glgC- were consistent with the proposed order. A simultaneous and approximately equivalent derepression of the glgC, glgA, and glgB (branching enzyme) gene products was observed in the late logarithmic-early stationary phase of growth on enriched media. These results are consistent with the coordinately regulated synthesis of the three glycogen biosynthetic enzymes in Salmonella typhimurium.

Genes↗
PubMed1977

[A mathematical model of the pyruvate oxidation in liver mitochondria. 1. Regulation of the Krebs cycle by adenine and pyridine nucleotides].

A mathematical model is proposed to describe the behavior of the pyruvate metabolic reactions, Krebs cycle and oxidative phosphorylation over a wide range of changes in the pyruvate influx rate and the activities of ATPase and NADH-reoxidating dehydrogenase. The role of adenine and pyridine nucleotides in various allosteric regulations of the Krebs cycle enzymes is discussed. The accumulation of ATP and NADH has been shown to proceed in definite succession, which makes the allosteric regulation of the Krebs cycle enzymes successive too. First "works" the inhibition by ATP, then by NADH. It has been shown that the properties of the model are in qualitative agreement with the experimental data (Garber A., Hanson R. [1]) on pyruvate oxidation by mitochondria from guinea pig liver, when allosteric regulation of isocitrate dehydrogenase by adenine nucleotides is taken into account.

Adenine Nucleotides↗
PubMedDate not supplied

[Comparative theoretic analysis of an open reaction S1 goes to and comes from S2 E(R,T) in which the oligomeric enzyme E(R,T) is isosterically or allosterically activated by the product S2].

In connection with evolutionary aspects of the mechanisms of allosteric regulation of cell metabolism, a mathematical model of an open reaction leads to S1 E(R,T) in equilibrium S2 leads to involving product activation of the olygomeric enzyme E(R,T) whose protomers undergo the concerted conformational transitions R in equilibrium T, has been analysed. Two activation mechanisms, isosteric and allosteric, were considered. Both mechanisms produce qualitatively the same effects: the input characteristic of the reaction possesses hysteresis, which causes multiple steady states and self-oscillations. In the case of isosteric activation, only a small fraction of the maximum enzyme activity is utilized because of the strong competition between S1 and S2 for the active sites. In the case of allosteric regulation, this competition may almost completely be eliminated and the enzyme activity may be utilized with high efficiency, provided the affinity of S2 for the allosteric sites is at least an order of magnitude higher than for the active sites. Qualitative similarity between the two cases of product activation, in spite of their great quantitative discrepancy, favours the hypothesis that the mechanisms of isosteric regulation were the direct precursors of the homological allosteric regulatory mechanisms.

Allosteric Regulation↗
PubMed1976

Heart phosphofructokinase: allosteric kinetics with fructose 6-sulfate.

The allosteric regulation of heart phosphofructokinase was studied at pH 6.9 with an alternative substrate, fructose 6-sulfate. The alternative substrate allowed kinetic studies to be carried out at high enzyme concentrations (0.1 mg/ml) where the effect of allosteric ligands on enzyme physical structure has been studied. A Km for ATP binding (8-10 muM) in the presence of saturating AMP concentrations was found which agreed well with the value obtained at pH 8.2, ATP inhibitory effects closely followed saturation of its substrate site. Hill plots for ATP inhibition gave an interaction coefficient of 3.5 indicating cooperatively between at least four enzyme subunits. Neither AMP nor fructose 6-sulfate affected the cooperativity between the ATP inhibitory sites but only increased the inhibitory threshold. As the ATP concentration was increased from suboptimal to inhibitory levels, interaction coefficients for AMP and fructose 6-sulfate changed from 1 to 2. Increasing citrate concentration resulted in an increase in the interaction coefficient for fructose 6-sulfate to a value of 1.9. Citrate inhibition was synergistic with ATP inhibition with an interaction coefficient of 2. The data indicate that allosteric kinetics of the enzyme can be shown at high enzyme concentrations with the alternative substrate. ATP inhibition appears to involve interaction between at least four subunits, while citrate, AMP, and fructose 6-sulfate interact minimally with two subunits.

Adenosine Monophosphate↗
PubMed1976

Deoxyribonucleotide pools in mouse-fibroblast cell lines with altered ribonucleotide reductase.

Mutant cells lines of 3T6 mouse fibroblasts, resistant to thymidine and deoxyadenosine, have an altered allosteric regulation of the enzyme ribonucleotide reductase (Meuth, M. and Green, H., Cell, 3, 367, 1974). Compared to 3T6, these lines contain larger pools of deoxynucleoside triphosphates, in particular deoxycytidine triphosphate, but show a normal rate of DNA synthesis. Addition of thymidine or deoxyadenosine to 3T6 cells results in large accumulations of the corresponding triphosphates and a dramatic decrease in the dCTP pool, concomitant with inhibition of DNA synthesis. Addition of thymidine to the mutant cell lines also leads to an increase in the dTTP pool but does not result in a depletion of dCTP or inhibition of DNA synthesis. Addition of deoxyadenosine only leads to a small increase of the dATP pool. In general the change in the allosteric regulation of bibonucleotide reductase is reflected in the deoxynucleotide pools.

Animals↗
PubMed1979

[Kinetic and allosteric properties of L-threonine-L-serine dehydratase from human liver].

It was shown that low concentrations of ATP (1..10(-4)M) and 10-fold concentrations of AMP (1.10(-3)M) at three constant L-threonine concentrations activated the L-threonine dehydratase activity of L-threonine-L-serine dehydratase from human liver, but had no effect on the L-serine dehydratase activity of this enzyme. Higher concentrations of both nucleotides inhibited the enzyme. The effects of ATP and AMP were specific. The activating and inhibiting effects of various concentrations of ATP and AMP were revealed as changes in the shapes of the curves for the initial reaction rate of the L-threonine dehydratase reaction versus initial substrate concentration. For this reaction the curves were not hyperbolic and were characterized by intermediary plateaux. ATP and AMP also influenced the maximal rate of the enzymatic reaction. Using the desensitization method it was shown that the activating effects of ATP and AMP are of allosteric nature. Thus, human liver L-threonine-L-serine dehydratase is an allosteric enzyme, for which positive allosteric effectors are low concentrations of ATP and AMP and negative allosteric effectors are high concentrations of these nucleotides. A possible mechanism of allosteric regulation of the enzyme under catalysis of the L-threonine dehydratase reaction and the lack of regulation under catalysis of the L-serine dehydratase reaction as well as specificity of the allosteric sites of this enzyme to the two nucleotides and the physiological significance of this process are discussed.

Adenosine Monophosphate↗
PubMedDate not supplied

[Description of the ratios between metabolite concentrations in a polyenzyme system].

It was demonstrated that the relations between substrate and product concentrations for a reaction catalyzed by michaelian enzyme incorporated in a multienzyme system can be graphically represented by a diverging set of straight lines intersecting in one point, the flux velocity being treated as a parameter. A competitive inhibitor shifts the intersection point along the line of equilibrium state. The relations between the concentrations of more than two reagents are represented by a set of equivelocity surfaces. The relations between substrate and product concentrations for a kinetically cooperative reaction conforming to the graphical representation by the second--order curves were analyzed. The stability criterion was obtained for a multienzyme system with the first enzyme allosterically regulated by products of subsequent reactions.

Allosteric Regulation↗