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PubMed2003

Allosteric regulation of Bacillus subtilis NAD kinase by quinolinic acid.

NADP is essential for biosynthetic pathways, energy, and signal transduction. In living organisms, NADP biosynthesis proceeds through the phosphorylation of NAD with a reaction catalyzed by NAD kinase. We expressed, purified, and characterized Bacillus subtilis NAD kinase. This enzyme represents a new member of the inorganic polyphosphate [poly(P)]/ATP NAD kinase subfamily, as it can use poly(P), ATP, or other nucleoside triphosphates as phosphoryl donors. NAD kinase showed marked positive cooperativity for the substrates ATP and poly(P) and was inhibited by its product, NADP, suggesting that the enzyme plays a major regulatory role in NADP biosynthesis. We discovered that quinolinic acid, a central metabolite in NAD(P) biosynthesis, behaved like a strong allosteric activator for the enzyme. Therefore, we propose that NAD kinase is a key enzyme for both NADP metabolism and quinolinic acid metabolism.

Allosteric Regulation↗
PubMed1998

Allosteric regulation of vaccinia virus ribonucleotide reductase, analyzed by simultaneous monitoring of its four activities.

As determined by simultaneous monitoring of its four activities, vaccinia virus-coded ribonucleoside diphosphate (rNDP) reductase shows responses to individual nucleoside triphosphate effectors-ATP, dATP, dGTP, and dTTP-similar to those previously reported for rNDP reductase of mouse, which the viral enzyme closely resembles. This investigation uses the vaccinia enzyme as a readily available and convenient model for understanding the cellular enzyme. As previously reported for T4 phage aerobic rNDP reductase, we found the relative activities of ADP, CDP, GDP, and UDP reduction to be reasonably close to the proportions of the four deoxyribonucleotides in the vaccinia virus genome, but only when the four substrates and the four allosteric effectors were all provided at their approximate intracellular concentrations. GDP reductase levels were somewhat higher, proportionately, than the representation of dGMP in vaccinia virus DNA. To understand this behavior and also to evaluate possible relationships between ribonucleotide reductase control and the very low dGTP pools seen in eukaryotic cells, we carried out substrate saturation experiments with a "bioproportional" mixture containing the four rNDP substrates at their relative in vivo concentrations as determined from rNDP pool measurements. Reduction of the two purine substrates was inhibited at high concentrations of this mixture, and data suggest that ADP acts as a specific inhibitor of its own reduction and that of GDP. Use of the four-substrate assay revealed also that a mixture of vaccinia virus R1 protein and mouse R2 protein is catalytically active, making this the first reported chimeric rNDP reductase to show biological activity.

Adenosine Triphosphate↗
PubMed1983

Allosteric regulation of the 16 alpha-hydroxylation of progesterone as catalyzed by rabbit microsomal cytochrome P-450 3b.

A variety of compounds that can arise from the metabolism of progesterone in vivo stimulate the 16 alpha-hydroxylation of progesterone as catalyzed by highly purified, reconstituted preparations of cytochrome P-450 3b obtained from rabbit strain IIIVO/J. In general, reduction of the 20-keto moiety of progesterone or related compounds increases the extent of stimulation. Reduction of the 3-keto group also results in increased stimulatory activity in most cases. The resulting 3 beta-hydroxy derivatives are more active than the corresponding 3 alpha-isomers. In a similar fashion, 5 beta-pregnanes exhibit greater activity than the corresponding 5 alpha-pregnanes. The effect of these allosteric effectors is saturable at relatively low concentrations when compared to other positive effectors of P-450-mediated metabolism. These compounds increase the apparent ratio of Vmax/Km without altering the amount of reductase required for half-maximal activity when reconstituted with the cytochrome. In contrast, many of these compounds do not affect or inhibit the 6 beta-hydroxylation of progesterone catalyzed by a subform of P-450 3b that is expressed in New Zealand White rabbits but not in strain IIIVO/J. Many of the compounds investigated here are metabolites of progesterone and, therefore, may modulate P-450 3b-mediated metabolism during pregnancy.

Allosteric Regulation↗
PubMed1995

Allosteric regulation of [3H]vinblastine binding to P-glycoprotein of MCF-7 ADR cells by dexniguldipine.

Plasma membranes were prepared from the P-glycoprotein expressing human breast cancer cell line MCF-7 ADR. [3H]Vinblastine bound to these membranes saturably with a Bmax of 24 pmol/mg of protein and KD of 23 nM. In contrast, membranes from the parent cells MCF-7 WT, which do not express P-glycoprotein, did not bind [3H]vinblastine with high affinity. Cytotoxics known to be transported by P-glycoprotein inhibited the binding of [3H]vinblastine, as did multidrug reversing agents including the 1,4-dihydropyridine, dexniguldipine-HCl (Ki, 15 nM). In dissociation kinetic experiments, dexniguldipine-HCl accelerated the dissociation of [3H]vinblastine from P-glycoprotein, indicating a negative heterotropic allosteric mechanism of action through a drug binding site distinct from that of vinblastine. Other 1,4-dihydropyridines tested also accelerated [3H]vinblastine dissociation from P-glycoprotein, however, multidrug reversing drugs of different chemical classes, including quinidine, verapamil and cyclosporin A did not. These results suggest that P-glycoprotein of MCF-7 ADR cell membranes possesses at least two drug acceptor sites which are allosterically coupled: receptor site-1 which binds vinca alkaloids, and receptor site-2 which binds 1,4-dihydropyridines such as dexniguldipine-HCl, which had the highest affinity of the tested derivatives.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Degradation of L-glutamate dehydrogenase from Escherichia coli: allosteric regulation of enzyme stability.

L-glutamate dehydrogenase (GDH) is stable in exponentially growing Escherichia coli cells but is degraded at a rate of 20-30% per hour in cells starved for either nitrogen or carbon. GDH degradation is energy-dependent, and mutations in ATP-dependent proteases, ClpAP or Lon lead to partial stabilization. Degradation is inhibited by chloramphenicol and is completely blocked in relA mutant cells, suggesting that ribosome-mediated signaling may facilitate GDH degradation. Purified GDH has a single tight site for NADPH binding. Binding of NADPH in the absence of other ligands leads to destabilization of the enzyme. NADPH-induced instability and sensitivity to proteolysis is reversed by tri- and dicarboxylic acids or nucleoside di- and triphosphates. GTP and ppGpp bind to GDH at an allosteric site and reverse the destabilizing effects of NADPH. Native GDH is resistant to degradation by several purified ATP-dependent proteases: ClpAP, ClpXP, Lon, and ClpYQ, but denatured GDH is degraded by ClpAP. Our results suggest that, in vivo, GDH is sensitized to proteases by loss of a stabilizing ligand or interaction with an destabilizing metabolite that accumulates in starving cells, and that any of several ATP-dependent proteases degrade the sensitized protein.

ATP-Dependent Proteases↗
PubMed1995

Allosteric regulation of carbamoylphosphate synthetase-aspartate transcarbamylase multifunctional protein of Saccharomyces cerevisiae: selection, mapping and identification of missense mutations define three regions involved in feedback inhibition by UTP.

The positive screening procedure previously described was used in order to select, clone and characterize mutants defective in negative feedback control by UTP of the yeast carbamoylphosphate synthetase-aspartate transcarbamylase protein (CPSase-ATCase). The selection procedure was improved by adding a general mapping method for dominant mutations in order to avoid sequencing the whole URA2 allele (7 kb). All 16 mutants obtained carry missense mutations leading to single amino acid replacements: five of them are located in the CPSase domain while the other 11 are in the ATCase domain. In these 16 mutants, ATCase is no longer inhibited by UTP although CPSase retains full sensitivity to the effector, suggesting that the regulation of the two activities involve distinct mechanisms. Amino acid replacements in the ATCase domain were located on a three-dimensional model structure of the yeast ATCase domain. They are clustered in two regions of this domain which must be directly involved in the feedback process.

Allosteric Regulation↗
PubMed2002

Allosteric regulation of mouse brain serine racemase.

Serine racemase, purified from mouse brain, consisted of two isoforms. They had similar enzymatic properties and had molecular weights of about 55 kDa based on size exclusion chromatography. This is about twice that reported from its electrophoretic mobility on SDS gels or from the amino acid sequence of the recombinant enzyme. In addition to the previously reported requirements for pyridoxal phosphate and reducing agents, we found that both forms of the enzyme required Mg2+ and were strongly stimulated by yeast extract. The yeast extract could be replaced by ATP, GTP, or ADP and, to a lesser extent, by other nucleotides. In the presence of 1 mM ATP, the Km for L-serine decreased from 13 mM to 1.8 mM with little change in Vmax, indicating an allosteric mechanism for nucleotide activation. In addition to acting as a serine racemase, the enzyme has been reported to act on L-serine O-sulfate as a dehydratase. When measured by HPLC, after derivatization with 2,4 dinitrophenylhydrazine, we found, as expected, a very rapid formation of pyruvate from this substrate. L-serine was also converted to pyruvate at about twice the racemization rate. L-serine O-sulfate dehydration was inhibited by ATP, while L-serine dehydration, like racemization, was activated by nucleotides, indicating that, for L-serine, dehydration and racemization take place at the same site.

Allosteric Regulation↗
PubMed1985

A paradox: the 5-HT2-receptor antagonist ketanserin restores the 5-HT-induced contraction depressed by methysergide in large coronary arteries of calf. Allosteric regulation of 5-HT2-receptors.

Methysergide depresses the contractile effects of 5-hydroxytryptamine (5-HT) in bovine large coronary arteries devoid of endothelium. The IC50 of methysergide for depression of the response to 5-HT was (-log mol/l) 9.8. A low sensitivity contractile effect of 5-HT was not influenced by 1-1,000 nmol/l methysergide. The maximum force of this residual response is approximately 1/3 of the maximum force elicited by 5-HT in the absence of methysergide. Ketanserin restored the 5-HT-induced contraction depressed by methysergide. In the presence of 0.1 mumol/l ketanserin, methysergide caused depression of the 5-HT-induced effects with an IC50 (-log mol/l) of 6.5 without affecting the residual response. We propose that methysergide depresses 5-HT-induced contractions by acting on an allosteric site. The effect of binding of methysergide to the allosteric site would lead to a conformational change of the 5-HT2-receptor, thereby only allowing the production of a residual 5-HT-induced contraction. Ketanserin competes with high affinity not only with 5-HT for the 5-HT2-receptor but also with methysergide for the allosteric site, thus shifting the receptor back into its original conformation. The affinity estimate of ketanserin for the allosteric site yielded a KB (-log mol/l) of 10.3. Ketanserin (1-1,000 nmol/l) antagonized the contractile effects of 5-HT with a potency expected from its affinity for 5-HT2-receptors (-log KB, mol/l 9.4). However, micromolar concentrations of ketanserin antagonized the effects of 5-HT less than expected from its affinity for 5-HT2-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed2001

Allosteric regulation of the cAMP receptor protein.

The cyclic AMP receptor protein (CRP) of Escherichia coli is a dimer made up of identical subunits. Each CRP subunit contains a cyclic nucleotide binding pocket and the CRP dimer exhibits negative cooperativity in binding cAMP. In solutions containing cAMP, CRP undergoes sequential conformation changes from the inactive apo-form through the active CRP:(cAMP)(1) complex to the less active CRP:(cAMP)(2) complex depending on the cAMP concentration. Apo-CRP binds DNA with low affinity and no apparent sequence specificity. The CRP:(cAMP)(1) complex exhibits high affinity, sequence-specific DNA binding and interacts with RNA polymerase, whether free in solution or complexed with DNA. The results of genetic, biochemical and biophysical studies have helped to uncover many of the details of cAMP-mediated allosteric control over CRP conformation and activity as a transcription factor. These studies indicate that cAMP binding produces only small, but significant, changes in CRP structure; changes that include subunit realignment and concerted motion of the secondary structure elements within the C-terminal DNA binding domain of each subunit. These adjustments promote CRP surface-patch interaction with RNA polymerase and protrusion of the F-helix to promote CRP site-specific interaction with DNA. Interactions between CRP and RNA polymerase at CRP-dependent promoters produce active ternary transcription complexes.

Allosteric Regulation↗
PubMed2002

Allosteric regulation of estrogen receptor structure, function, and coactivator recruitment by different estrogen response elements.

Hormone-activated ERs (ERalpha and ERbeta) bind with high affinity to specific DNA sequences, estrogen response elements (EREs), located within the regulatory regions of target genes. Once considered to function solely as receptor tethers, there is an increasing amount of recent evidence to suggest that the sequence of the ERE can influence receptor activity. In this study, we have performed a systematic analysis of the role of different EREs in ER pharmacology. Specifically, by measuring ER activity on the vitellogenin A2, complement 3 gene, pS2, and lactoferrin EREs, we demonstrate that the activities of E2 and xenoestrogen ligands through ERalpha and ERbeta are significantly influenced by the nature of the response element. Using a series of ERalpha and ERbeta interacting peptides that contain the coactivator-binding motif LXXLL, we show that the type of ERE with which the receptor associates regulates the structure of the coactivator pocket on ER. Furthermore, using a novel ELISA developed to measure ER-coactivator interactions revealed that these different conformational states of ERalpha and ERbeta are functionally relevant, as they dictate receptor coactivator binding preference. Together, these results indicate that the DNA response element is a key regulator of receptor structure and biological activity and suggest the ERE sequence influences the recruitment of coactivators to the ER at target gene promoters. We propose that DNA-induced alteration of protein structure and coregulator recruitment may serve as a universal regulatory component for differential gene expression by other nuclear hormone receptors and unrelated transcription factors.

Allosteric Regulation↗
PubMed2006

Protein-protein interactions in the allosteric regulation of protein kinases.

Protein-protein interactions involving the catalytic domain of protein kinases are likely to be generally important in the regulation of signal transduction pathways, but are rather sparsely represented in crystal structures. Recently determined structures of the kinase domains of the mitogen-activated protein kinase Fus3, the RNA-dependent kinase PKR, the epidermal growth factor receptor and Ca(2+)/calmodulin-dependent protein kinase II have revealed unexpected and distinct mechanisms by which interactions with the catalytic domain can modulate kinase activity.

Allosteric Regulation↗
PubMed1990

[Allosteric regulators of reversible oxygenation of hemoglobin].

Properties of regulators of reversible haemoglobin oxygenation, especially of a natural regulator, 2,3-diphosphoglyceric acid (DPG), are reviewed. DPG provides effective deoxygenation of haemoglobin, helps to maintain its functional properties preventing its transformation into inactive derivatives. Correlation between organism metabolic requirements and haemoglobin oxygen affinity is established by erythrocyte DPG. Several functional analogues of DPG, their structure--activity relationship and possible medical application are discussed.

2,3-Diphosphoglycerate↗
PubMed1999

Allosteric regulation of even-skipped repression activity by phosphorylation.

The Drosophila homeodomain protein Even-skipped (Eve) is a well characterized transcriptional repressor. Here, we show that Eve's ability to function in vitro is negatively regulated by phosphorylation. DNA-binding activity was unaffected by phosphorylation, but phosphorylated Eve was unable to interact with the TATA-binding protein (TBP), a known target for repression. Unexpectedly, phosphorylation of the Eve N terminus, which is dispensable for repression and TBP binding, was necessary and sufficient to inactivate Eve. LiCl, which specifically inhibits glycogen synthase kinase-3 (GSK-3), reduced Eve phosphorylation in nuclear extract and blocked inhibition of repression. In addition, Eve was phosphorylated and inactivated by purified GSK-3 beta plus casein kinase II. Our results suggest a novel mechanism of transcriptional control involving phosphorylation-induced allosteric interference with a repressive protein-protein interaction.

Allosteric Regulation↗
PubMed2001

Kinetic mechanism of allosteric regulation of muscle glycogen phosphorylase B by adenosine 5'-monophosphate.

Kinetic analysis of the glycogen chain growth reaction catalyzed by glycogen phosphorylase b from rabbit skeletal muscle has been carried out over a wide range of AMP concentration under the saturation of the enzyme by glycogen. Applicability of some variants of the kinetic model involving the interaction of AMP- and glucose 1-phosphate-binding sites in the dimeric enzyme molecule is considered. A kinetic model of the enzymatic reaction describing adequately the activation of the enzyme by AMP and inhibition at sufficiently high concentrations of AMP is proposed.

Adenosine Monophosphate↗
PubMed1997

Unique allosteric regulation of 5-hydroxytryptamine receptor-mediated signal transduction by oleamide.

The effects of oleamide, an amidated lipid isolated from the cerebrospinal fluid of sleep-deprived cats, on serotonin receptor-mediated responses were investigated in cultured mammalian cells. In rat P11 cells, which endogenously express the 5-hydroxytryptamine2A (5HT2A) receptor, oleamide significantly potentiated 5HT-induced phosphoinositide hydrolysis. In HeLa cells expressing the 5HT7 receptor subtype, oleamide caused a concentration-dependent increase in cAMP accumulation but with lower efficacy than that observed by 5HT. This effect was not observed in untransfected HeLa cells. Clozapine did not prevent the increase in cAMP elicited by oleamide, and ketanserin caused an approximately 65% decrease. In the presence of 5HT, oleamide had the opposite effect on cAMP, causing insurmountable antagonism of the concentration-effect curve to 5HT, but had no effect on cAMP levels elicited by isoproterenol or forskolin. These results indicate that oleamide can modulate 5HT-mediated signal transduction at different subtypes of mammalian 5HT receptors. Additionally, our data indicate that oleamide acts at an apparent allosteric site on the 5HT7 receptor and elicits functional responses via activation of this site. This represents a unique mechanism of activation for 5HT G protein-coupled receptors and suggests that G protein-coupled neurotransmitter receptors may act like their iontropic counterparts (i.e., gamma-aminobutyric acid type A receptors) in that there may be several binding sites on the receptor that regulate functional activity with varying efficacies.

Allosteric Regulation↗
PubMed1999

A mutation that uncouples allosteric regulation of carbamyl phosphate synthetase in Drosophila.

In animals, UTP feedback inhibition of carbamyl phosphate synthetase II (CPSase) controls pyrimidine biosynthesis. Suppressor of black (Su(b) or rSu(b)) mutants of Drosophila melanogaster have elevated pyrimidine pools, and this mutation has been mapped to the rudimentary locus. We report that rSu(b) is a missense mutation resulting in a glutamate to lysine substitution within the second ATP binding site (i.e. CPS.B2 domain) of CPSase. This residue corresponds to Glu780 in the Escherichia coli enzyme (Glu1153 in hamster CAD) and is universally conserved among CPSases. When a transgene expressing the Glu-->Lys substitution was introduced into Drosophila lines homozygous for the black mutation, the resulting flies exhibited the Su(b) phenotype. Partially purified CPSase from rSu(b) and transgenic flies carrying this substitution exhibited a dramatic reduction in UTP feedback inhibition. The slight UTP inhibition observed with the Su(b) enzyme in vitro was due mainly to chelation of Mg2+ by UTP. However, the Km values for glutamate, bicarbonate, and ATP obtained from the Su(b) enzyme were not significantly different from wild-type values. From these experiments, we conclude that this residue plays an essential role in the UTP allosteric response, probably in propagating the response between the effector binding site and the ATP binding site. This is the first CPSase mutation found to abolish feedback inhibition without significantly affecting other enzyme catalytic parameters.

Adenosine Triphosphate↗
PubMed1995

Allosteric regulation of proton translocation by a vacuolar adenosinetriphosphatase.

The kinetics of nucleoside-triphosphate-dependent proton translocation by a vacuolar-type adenosine-triphosphatase have been studied, using the enzyme from bovine chromaffin-granule membranes, purified and reconstituted into proteoliposomes. The reaction was followed by recording the quenching of the fluorescence of the permeant weak base 9-amino-6-chloro-2-methoxyacridine; fluorescence data were collected and stored in digital form, and the initial reaction rates estimated by linear regression. In the absence of nucleoside diphosphate, the dependence of initial rates of proton translocation on substrate concentration were fitted well by the Michaelis-Menten equation, as were the kinetics of ATP hydrolysis. ADP and other nucleoside diphosphates were potent inhibitors of the ATPase, effecting a reduction in the maximum velocity of the reaction, and producing sigmoid substrate-saturation curves which could be fitted by the empirical Hill equation, the Hill coefficient approaching 2 at high inhibitor concentrations. Data sets containing initial-rate estimates were collected over a wide range of independently varied concentrations of substrate and inhibitor and were modeled, using rate equations derived from several different models based on the concerted-transition model of allosteric inhibition proposed by Monod, Wyman and Changeux. These equations were fitted to the data by weighted non-linear regression, using an iterative computer program to obtain the best estimates of kinetic parameters. One model consistently fitted all of the data sets better than all the others, and this model was based on the following assumptions: that the ATPase exists in two conformational states, R and T; that only the R state is catalytically active; that each state contains three kinetically equivalent catalytic sites, and one regulatory site; that nucleoside triphosphates bind only to the catalytic sites, and that nucleoside diphosphates bind both to the catalytic sites and to the regulatory site. The optimized values of the kinetic parameters indicate that in the absence of nucleoside diphosphate, the enzyme is almost completely in the R state; that nucleoside triphosphates bind more tightly to the R than to the T state; that binding of nucleoside diphosphates to the regulatory site is very tight, but occurs only in the T state; and that competitive binding of nucleoside diphosphates at the catalytic sites is stronger in the T state than in the R state. Experiments conducted with varying total magnesium concentrations indicated that the magnesium complexes of nucleoside diphosphates are much stronger inhibitors than the free nucleotides, and that free nucleoside triphosphates are weakly inhibitory, probably competing with the magnesium complexes for binding at the catalytic sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1983

Allosteric regulation of the NAD malic enzyme from cauliflower: activation by fumarate and coenzyme A.

Activation of the NAD malic enzyme is shown to be caused by free, uncomplexed fumarate2-. Mg-fumarate has no detectable effect on the enzyme. Fumarate2- isotherms are biphasic in that they consist of an activating as well as a deactivating region. Activation is shown to result from an increase in the affinity of the enzyme for malate2- while deactivation results from a reduction in Vmax. Phosphate does not affect the response of the enzyme to fumarate2-, while Cl- inhibits the enzyme in a manner that cannot be overcome by fumarate2-. SO42-, another activator of the malic enzyme, reduces the Ka for fumarate2- from 3.9 to 2.1 mM. Activation of the enzyme by coenzyme A (CoA) is hyperbolic with a Ka for CoA of 2.1 microM. Fumarate2- reduces this value to 1.2 microM. CoA, like SO42-, is able to increase the affinity of the enzyme for fumarate2-, decreasing its Ka by 56%. An additional effect of fumarate2- is to cause the interconversion of different catalytic forms of the enzyme which exist when Mg2- is limiting. On the basis of these results, a model of the number and types of allosteric sites present on the NAD malic enzyme is proposed.

Allosteric Regulation↗