Concerted Inhibition Research Articles

Regulation of isocitrate metabolism in Chlamydomonas segnis

Isocitrate lyase (EC 4.1.3.1) and isocitrate dehydrogenase (NADP+) (EC 1.1.1.42) activities were detected in cell-free extracts of Chlamydomonas segnis Ettl when the alga was grown photoautotrophically with 5% CO2 in air (v/v) at 11 klx. When the cultures were either bubbled with air (0.03% CO2), exposed to low light intensity (3 klx), or subjected to manganese or nitrogen deficiency, isocitrate lyase activity was undetectable. During growth in batch cultures provided with 5% CO2, the activity of the dehydrogenase was about 5–12 times greater than the lyase.Using partially purified (about 50-fold) enzyme preparations, isocitrate dehydrogenase (NADP+) showed greater affinity for isocitrate (Km = 0.008 mM) than did isocitrate lyase (Km = 0.1 mM). The dehydrogenase had Km values of 0.011 mM and 0.006 mM for NADP and Mn2+, respectively. Both enzymes were inhibited by α-ketoglutarate and oxalacetate at 1 mM, but the dehydrogenase was more sensitive to these two keto acids (68–79%) than the lyase (36%). Glycolate at 1 mM inhibited (36%) only the lyase, while glyoxylate had little effect. The dehydrogenase was subject to concerted inhibition by oxalacetate plus glyoxylate (Ki = 0.01 mM). This inhibition was competitive with respect to isocitrate, and preincubation of the enzyme with NADP in absence of isocitrate was necessary for effective inhibition. Each of NADPH (Ki = 0.06 mM) and ATP (Ki = 0.65 mM) was a non-competitive inhibitor (with respect to isocitrate) of isocitrate dehydrogenase (NADP+), and both nucleotides are suggested to be active in the in vivo regulation of isocitrate metabolism in C. segnis during photoautotrophy.

Multiple basis of combination chemotherapy

In combination chemotherapy, the type of drug interactions can be divided into three broad categories: 1) combinations based on cooperative effects of active drugs; 2) combinations in which the effectiveness of an active drug is increased by the concurrent administration of an inactive agent; and 3) combination of an active drug with an agent capable of selectively reversing the toxicity of the first drug. Many concepts have been proposed to explain the synergistic interaction between two active drugs at the level of the target cell. These include multiple inhibition of a single enzyme, enhanced activation, decreased inactivation, increased drug uptake, sequential blockade, concurrent inhibition, complimentary inhibition, and concerted inhibition. The therapeutic advantage of combination chemotherapy may reside in the whole organism, reflecting increased bioavailability of drug, reduced dose-limiting toxicity or reduced impairment of host defenses; it may reside in the tumor cells, reflecting the multiple molecular mechanisms of interaction mentioned above. Examples discussed include among others methotrexate plus citrovorum factor, thymidine or allopurinol, araC plus tetrahydrouridine and 3-deazauridine plus testosterone.

Concerted inhibition of NADP +-specific isocitrate dehydrogenase by oxalacetate and glyoxylate. I. Oxalomalate formation and stability, and nature of the enzyme inhibition

Oxalacetate and glyoxylate are each weak inhibitors of NADP +-specific isocitrate dehydrogenase ( threo- d S-isocitrate:NADP + oxidoreductase (decarboxylating), EC 1.1.1.42). Together, however, they act in a concerted manner and strongly inhibit the enzyme. The rates of formation and dissociation of the enzyme inhibitor complex, and the rate of formation and the stability of the aldol condensation product of oxalacetate and glyoxylate, oxalomalate, were examined. The data obtained do not support the often suggested possibility that oxalomalate, per se, formed non-enzymatically in isocitrate dehydrogenase assay mixtures containing oxalacetate and glyoxylate, is reponsible for the observed inhibition of the enzyme. Rather, the data presented in this communication suggest that oxalacetate binds to the enzyme first, and that the subsequent binding of glyoxylate leads to the formation of a catalytically inactive enzyme-inhibitor complex.

Studies on NADP<sup>+</sup>-specific Isocitrate Dehydrogenase from an Extreme Thermophile, <italic>Thermus flavus</italic> AT-62<xref ref-type="fn" rid="fn1"><sup>1</sup></xref><subtitle>Partial Purification and Properties</subtitle>

1. NADP-+-specific isocitrate dehydrogenase [EC 1.1.1.42] was partially purified by about 440-fold from an extreme thermophile, Thermus flavus AT-62. 2. Remarkable thermostability of the enzyme was confirmed. The enzyme was not inactivated after 60 min at 70 degrees, and the activity was lost only slowly at 80 degrees. Above 90 degrees, however, rapid inactivation was observed. 3. The dehydrogenase was susceptible to concerted inhibition by oxaloacetate plus glyoxylate. In the presence of oxaloacetate plus glyoxylate (each 1 mM), 75 percent inhibition was observed. 4. The degree of inhibition of the enzyme by oxaloacetate plus glyoxylate decreased markedly above 60 degrees. The affinity of the enzyme for isocitrate and NADP-+ was also reduced markedly above 60 degrees. The activation energy calculated from Arrhenius plots below and above 60 degrees were 14,500 and 8,000 cal per mole, respectively. These observations suggest a possible conformation change of the enzyme protein at a transition temperature of 60 degrees, and the physiological significance of this in the adaptation of thermophiles to elevated temperatures is discussed.

Concerted Feedback Inhibition: PURIFICATION AND SOME PROPERTIES OF ASPARTOKINASE FROM PSEUDOMONAS FLUORESCENS

Abstract Aspartokinase of Pseudomonas fluorescens has been purified by heat treatment, ammonium sulfate fractionation, DEAE-cellulose chromatography, and by successive gel filtration steps on Sephadex G-200 in the presence and absence of feedback modifiers. The 700-fold purified enzyme was judged to be greater than 90% pure. Sedimentation velocity centrifugation yielded an s020,w value of 6.7 S, a D20,w value of 5.3 x 10-7 cm2 s-1, and a molecular weight of 133,000. The Stokes radius of the native enzyme in 50 mm potassium phosphate buffer, pH 7.5, containing 200 mm KCl was found to be about 43 A. A diffusion coefficient of 5.2 x 10-7 cm2 s-1 was calculated from the Stokes-Einstein equation. A molecular weight of 137,000 was also estimated from the gel filtration data. The molecular weight of the subunit was 43,000. The enzyme had an absolute requirement for ATP and a divalent cation. No other nucleoside phosphates served as the phosphate donor. Addition of 200 mm KCl increased the reaction rate by about 40%; sodium and lithium ions had no effect. The activity of this enzyme was inhibited by concerted feedback by two pairs of amino acid end products: lysine plus threonine and methionine plus threonine. Individually, lysine and methionine were slightly stimulatory; threonine showed a slight activation at low concentrations, but was weakly inhibitory at concentrations over 10 mm. Concerted inhibition by low threonine and high lysine combination was compensated by methionine; whereas, methionine did not counteract when enzyme activity was severely decreased by high concentrations of threonine plus lysine. A possible physiological significance of the dual role of methionine is discussed.

Aspartokinase from Wheat Germ

Aspartokinase has been isolated from wheat germ and a preliminary survey made of its properties in a partially purified extract. The enzyme has an absolute requirement for ATP and a divalent metal ion. The phosphate donor can be either ATP or GTP, but other nucleotides are ineffective. Both magnesium and manganese will activate the enzyme, whereas calcium shows a trace amount of activity. The enzyme has a Km of 16.7 mm for aspartate, 1.2 mm for ATP, and 3.3 mm for MgCl(2). Lysine inhibits the reaction at fairly low concentrations, and threonine inhibits at high concentrations. Other amino acids which are derived from aspartate (methionine, homoserine, threonine, and isoleucine) have little effect. When lysine and threonine are added together, they show a concerted inhibition of the reaction. The enzyme is also stabilized against heat inactivation by lysine and threonine together but not by either when added separately. It is suggested that aspartokinase from plants is a regulatory enzyme and exhibits a concerted feedback mechanism.

Aspartokinase in Lemna minor L

The growth of Lemna minor was followed by means of frond number, fresh weight, and dry weight measurements in the presence of various amino acids at a concentration 0.25 mm. Lysine inhibited growth but not to the same extent as threonine and homoserine. Isoleucine was also an inhibitor of growth. In the presence of methionine there was some growth for 2 to 3 days, but by 5 days most of the plants appeared to be dead. When lysine and threonine were added together, there was no growth at all, and the plants were dead after 5 days. This effect of lysine + threonine could be reversed by adding methionine or homoserine to the growth medium.The isolated aspartokinase from Lemna showed inhibition by lysine and higher concentrations of threonine. When these amino acids were added together at low concentrations, there was a concerted inhibition of the aspartokinase. It is suggested that some effects of amino acids on the growth of L. minor can be explained on the basis of a concerted feedback control of aspartokinase.

Concerted feedback inhibition of the aspartokinase of Rhodospirillum tenue by threonine and methionine: a novel pattern.

The presence of a single aspartokinase was demonstrated in Rhodospirillum tenue. The enzyme has been purified about 60-fold. No physical association exists in this species between aspartokinase and homoserine dehydrogenase. The general properties of the enzyme are described. Inhibition by l-lysine, by l-threonine, and concerted inhibition by these two end products are regulatory characters which have also been found in many other species. In R. tenue, aspartokinase is also subject to a hitherto not encountered type of concerted feedback inhibition, by l-threonine plus l-methionine. The inhibition caused by lysine can be reversed either by glycine, l-isoleucine, l-methionine, or l-phenylalanine. The concerted inhibition by lysine plus threonine is reversed by glycine, l-isoleucine, or l-phenylalanine, but not by l-methionine, which exerts in conjunction with threonine the independent concerted inhibition referred to above. Addition of single or several metabolites to cultures of R. tenue caused inhibition of growth and reversal of growth inhibition, compatible with the effects observed in vitro on aspartokinase activity. The regulation of this enzyme in relation to that of other bacterial aspartokinases is discussed.

Regulation of isocitrate metabolism in peroxisomes in Tetrahymena pyriformis

Isocitrate dehydrogenase (NADP +-dependent) and isocitrate lyase compete for substrate in peroxisomes (microbodies) in the ciliated protozoan Tetrahymena pyriformis. Thus, some of the factors that might regulate the metabolism of isocitrate in Tetrahymena peroxisomes were studied. The dehydrogenase has a much greater affinity for isocitrate than does the lyase, but is subject to a concerted feedback inhibition by oxalacetate and glyoxylate. Concentrations of 25 μ m of each cause almost complete inhibition. The degree of inhibition is greatly reduced unless NADP is present during a brief preincubation of the enzyme with the inhibitors. Either glyoxylate or oxalacetate alone is also capable of inhibiting the enzyme, although higher concentrations are required. In this case, there is no inhibition unless inhibitor and enzyme are preincubated in the presence of NADP. Neither the concerted inhibition nor that due to oxalacetate seems to be competitive with isocitrate. Properties of the enzyme from the soluble fraction of the cell were identical to those of the peroxisomal enzyme. Enzyme from mouse liver exhibited most of the properties observed with the Tetrahymena enzyme. The concerted inhibition of isocitrate dehydrogenase may be the means by which isocitrate in the peroxisome is shunted through the glyoxylate bypass.

Sulfhydril groups and the concerted inhibition of NADP + -linked isocitrate dehydrogenase.

Mixtures of glyoxalate and oxaloacetate have been shown to inhibit highly purified pig heart NADP+ -linked isocitrate dehydrogenase in a strongly concerted manner. The α-ketoglutarate reductive carboxylase activity of the enzyme was also inhibited, but to a lesser extent than the dehydrogenase activity. Modification of sulfhydryl groups in the enzyme by certain reagents led to a diminution of the inhibition, although the extent of this effect depended on the reagent used. N-Ethylmaleimide was most effective at diminishing the concerted inhibition, whereas oxidation of the sulfhydryl groups by X-irradiation or lipid peroxide had no effect. It was concluded that the effect of sulfhydryl reagents on the inhibition was probably due to the modification of one specific cysteine residue which is possibly adjacent to the binding site on the enzyme for glyoxalate.

Regulation of aspartokinase activity in a thermophilic bacterium

The aspartokinase activity from the thermophilic organism studied ( I. Epstein and N. Grossowicz J. Bacteriol., 99 (1969) 414) is very sensitive to feedback inhibition by either L-threonine or L-lysine. Concerted inhibition was seen when both amino acids were added. This inhibition could be reversed by several amino acids, L-alanine and L-valine being most effective. Growth of this thermophilic organism was inhibited by addition of L-threonine to the medium. The growth inhibition could be reversed by the addition of L-methionine, L-homoserine or L-isoleucine.

Regulation of two aspartokinases in Bacillus subtilis.

When grown on minimal glucose medium, transformable Bacillus subtilis strains contained two distinct aspartokinases (ATP:l-aspartate 4-phosphotransferase, EC 2.7.2.4). One of these enzymes was inhibited by l-lysine (Lys), whereas the other was insensitive to inhibition but was activated by l-leucine. None of the other amino acids tested had any effect, and the addition of l-threonine did not enhance the inhibition by Lys, in contrast to the concerted inhibition observed for other bacilli. At the end of exponential growth, the Lys-sensitive aspartokinase activity decreased, whereas the Lys-insensitive activity remained relatively constant throughout the stationary phase. The two activities were separated by (NH(4))(2)SO(4) fractionation and Sephadex G-200 chromatography. Growth in the presence of Lys reduced the specific activity of aspartokinase by about 50% and eliminated the inhibition by Lys. In extracts of these cells, only Lys-insensitive activity was found upon (NH(4))(2)SO(4) fractionation and Sephadex G-200 chromatography. Lys apparently repressed the synthesis of the Lys-sensitive enzyme.

Studies on the Fermentative Production and Metabolism of Uridine Coenzymes

Enzyme activities involved in the galactose metabolism of Torulopsis candida grown on u lactose medium were investigated with the cell-free extract and ammonium sulfate fraction. Remarkable activities of galactokinase, galactose-l-phosphate uridylyltransferase and UDPG pyrophosphorylase were detected, whereas UDPGal pyrophosphorylase activity was weak. UDPGal formation proceeded by the cell-free extract along a coupling reaction catalyzed by UDPG pyrophosphorylase and galactose-l-phosphate uridylyltransferase where UDPG or glucose-l-phosphate acted as a catalyst. The mechanism of UDPGal accumulation under the fermentative condition could be explained by a concerted inhibition of UDPGal-4- epimerase activity by 5'-UMP and galactose present as fermentation substrates.

Nicotinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase from a higher plant. Isolation and charactertization.

Nicotinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase was extracted from etiolated pea (Pisum sativum L.) seedlings and was purified 65-fold. The purified enzyme exhibits one predominant protein band by polyacrylamide gel electrophoresis, which corresponds to the dehydrogenase activity as measured by the nitro blue tetrazolium technique. The reaction is readily reversible, the pH optima for the forward (nicotinamide adenine dinucleotide phosphate reduction) and reverse reactions being 8.4 and 6.0, respectively. The enzyme has different cofactor and inhibitor characteristics in the two directions. Manganese ions can be used as a cofactor for the reaction in each direction but magnesium ions only act as a cofactor in the forward reaction. Zinc ions, and to a lesser extent calcium ions, inhibit the enzyme at low concentrations when magnesium but not manganese is the metal activator. It is suggested that there is a fundamental difference between magnesium and manganese in the activation of the enzyme. The enzyme shows normal kinetics and the Michaelis contant for each substrate was determined. The inhibition by nucleotides, nucleosides, reaction products, and related compounds was studied. The enzyme shows a linear response to the mole fraction of reduced nicotinamide adenine dinucleotide phosphate when total nicotinamide adenine dinucleotide phosphate (nicotinamide adenine dinucleotide phosphate plus reduced nicotinamide adenine dinucleotide phosphate) is kept constant. Isocitrate in the presence of divalent metal ions will protect the enzyme from inactivation by p-chloromercuribenzoate. Protection is also afforded by manganese ions alone but not by magnesium ions alone There is a concerted inhibition of the enzyme by oxalacetate and glyoxylate.

Genetically desensitized aspartate kinase to the concerted feedback inhibition in Brevibacterium flavum.

Aspartate kinases [ATP: L-aspartate 4-phosphotransferase, EC 2. 7. 2. 4] were partially purified from Brevibacterium flavum and its mutant, which is resistant to L-threonine plus S-(2-aminoethyl)-L-cysteine (AEC), * a lysine analogue, and produces a large amount of L-lysine, and their properties were compared with each other. Both enzymes showed a homotropic interaction of the substrate, aspartate, which disappeared in the presence of the activator, threonine or ammonium sulfate. The degree of activation by ammonium sulfate was larger with the parental enzyme than with the mutant enzyme. In the presence of ammonium sulfate, double reciprocal plots of the reaction rate against one substrate concentration at various fixed concentrations of another substrate were linear and met at a point with both enzymes. Moreover, ADP, one of the reaction products inhibited the enzymes competitively to both substrates, aspartate and ATP, suggesting rapid equilibrium random Bi Bi mechanism for both enzymatic reactions. Kms for aspartate and ATP were similar with both enzymes. Threonine slightly activated the mutant enzyme but partially competitively inhibited the parental enzyme in the presence of ammonium sulfate, while, in the absence of the salt, it was an activator for both enzymes. Gel filtration experiments showed dimer formation by the addition of threonine in the presence of ammonium sulfate. Regardless of the presence of ammonium sulfate, lysine inhibited both enzymes to the same degree at a high concentration. In contrast to the parental enzyme, concerted inhibition by lysine plus threonine was not observed with the mutant enzyme. Furthermore, a simultaneous addition of threonine diminished the inhibitory effect of lysine. Isoleucine only slightly activated the mutant enzyme, while about twice activation was observed with the parental enzyme. In conclusion, a genetic alteration occurred in aspartate kinase of an analogue-resistant mutant affected all tested actions of the allosteric effectors, threonine, isoleucine, and ammonium sulfate but not those of the substrates and the competitive inhibitor, lysine. Effect of AEC was similar to those of lysine with both enzymes. Thus, the specific growth inhibition of the parental strain by this analogue plus threonine which was reversed by the addition of lysine, seems to be caused by the concerted inhibition of the aspartate kinase. The resistance of the mutant to these amino acids as well as lysine overproduction in the mutant can be well explained by the lack of the concerted inhibition in the mutant enzyme.

Alternative Allosteric Effects Exerted by End Products upon a Two-Substrate Enzyme in Rhodomicrobium vannielii

Abstract The regulatory enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthetase of Rhodomicrobium vannielii was studied. Both phenylalanine and tyrosine are competitive inhibitors with respect to the substrate, erythrose 4-phosphate. Tryptophan does not influence enzyme activity per se, but potentiates the effect of phenylalanine or tyrosine. Kinetic experiments led to the conclusion that alternative kinetic pathways to the ternary complex existed, and that the one forming (phosphoenolpyruvate-enzyme) as the initial binary complex was kinetically preferred. Accordingly, the ratio of the two substrates had a marked influence upon reaction velocity. The observed ability of phenylalanine and tyrosine to either activate or inhibit enzyme activity, depending upon the ratio of the two substrates was consistent with the postulated mechanism. An extremely wide range of enzyme activity exists, on the one hand, because of the stimulation of activity by one or two amino acid end products, and on the other hand, because of the potent concerted inhibition in the simultaneous presence of all three amino acids. The physiological consequences of this pattern of control for 3-deoxy-d-arabino-heptulosonate 7-phosphate synthetase are discussed.

Feedback Inhibition of an Allosteric Triphosphopyridine Nucleotide-specific Isocitrate Dehydrogenase

Abstract Evidence is presented for the concerted inhibition of a TPN+-specific isocitrate dehydrogenase from Crithidia fasciculata by two biosynthetic intermediates, oxalacetate and glyoxylate. This inhibition is competitive with substrate, and the presence of either inhibitory compound greatly increases the affinity of the enzyme for the second. This inhibition has been shown not to be due to the formation of oxalomalate, the nonenzymatic condensation product. Structural analogues of the substrate are not inhibitory, so that steric configurational analogy is apparently not the mechanism of inhibition. In view of this, it is believed that an alteration in the protein must be induced by the combination of the two inhibitors, as has been demonstrated previously with this enzyme for ATP. This work confirms the allosteric nature of the ATP inhibition and substantiates the requirement for 2 moles of ATP per mole of enzyme. Since isocitrate can either enter the glyoxylate cycle or be metabolized via the tricarboxylic acid cycle, it is believed that the inhibition of this enzyme by oxalacetate and glyoxylate has biological significance.

Regulation of isocitrate dehydrogenase from Thiobacillus thiooxidans and Pseudomonas fluorescens

The isocitrate dehydrogenases of T. thiooxidans and P. fluorescens have been studied. The apparent K M's for isocitrate are 1.2 × 10 −5 M and 1.5 × 10 −5 M respectively. The apparent K M for NAD + is 2.9 × 10 −4 M for the enzyme from T. thiooxidans while the apparent K M for the NADP-specific enzyme of P. fluorescens is 1.7 × 10 −5 M. ADP and ATP were found to inhibit the isocitrate dehydrogenases of both organisms. Glyoxalate plus oxalacetate caused concerted inhibition of the enzyme from T. thiooxidans. Glyoxalate, when added alone, was an activator of the enzyme from P. fluorescens but increased the inhibition due to oxalacetate and reduced the K I for oxalacetate by three orders of magnitude.

Concerted inhibition and its reversal by end products of aspartate kinase in Brevibacterium flavum.

Concerted inhibition and its reversal by end products of aspartate kinase in Brevibacterium flavum.

The regulation of aspartokinase in Bacillus licheniformis

The specific activity of aspartokinase (ATP: l-aspartate 4-phosphotransferase, EC 2.7.2.4) decreases rapidly to a 10 to 20-fold lower level at the end of the growth phase in Bacillus licheniformis grown on a minimal glucose-salts medium. Concurrent with this decrease in specific activity is a loss of sensitivity to feedback inhibition by l-lysine and a slower decrease in the concerted inhibition caused by l-lysine and l-threonine. Growth of the cells in the minimal medium plus l-lysine and l-threonine caused the production of aspartokinase with characteristics similar to those of enzyme from cells harvested 4 h after the end of growth. Growth in the presence of either l-lysine or l-threonine did not affect the specific activity of the enzyme but did alter its inhibition sensitivity. The addition of l-lysine to the medium during growth on the minimal medium caused a rapid loss of sensitivity to lysine inhibition, 50% desensitization occurring in 15 min. These latter conditions did not produce an alteration in the specific activity of the enzyme or in the concerted inhibition by lysine and threonine. Despite the marked alteration in feedback-inhibition properties, changes in physical or kinetic properties of purified aspartokinase were not observed. Throughout the purification steps there was no evidence that more than one aspartokinase was present in B. licheniformis cells. It is concluded that this enzyme is unusually plastic and assumes alternate forms depending on the physiology of the cell.