[Proceedings: Regulation by allosteric interaction. Regulatory properties of pyvate kinase in different tissues of mammals].
Explore the source record for details and available documents.
Allosteric Regulation↗
SEARCH · Search PubMed
Results for “Allosteric Regulation”
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Temperature-dependent effects of cadmium and purine nucleotides on mitochondrial aconitase from a marine ectotherm, Crassostrea virginica: a role of temperature in oxidative stress and allosteric enzyme regulation.
Temperature and heavy metals such as cadmium (Cd) are important environmental stressors that can strongly affect mitochondrial function of marine poikilotherms. In this study, we investigated the combined effects of temperature (20 degrees C and 30 degrees C) and Cd stress on production of reactive oxygen species (ROS) and oxidative stress in a marine poikilotherm Crassostrea virginica (the eastern oyster) using mitochondrial aconitase as a sensitive biomarker of oxidative damage. We also assessed potential involvement of mitochondrial uncoupling proteins (UCPs) in antioxidant protection in oyster mitochondria using purine nucleotides (GDP, ATP and ADP) as specific inhibitors, and free fatty acids as stimulators, of UCPs. Our results show that exposure to Cd results in elevated ROS production and oxidative damage as indicated by aconitase inactivation which is particularly pronounced at elevated temperature. Unexpectedly, oyster mitochondrial aconitase was inhibited by physiologically relevant levels of ATP (IC(50)=1.93 and 3.04 mmol l(-1) at 20 degrees C and 30 degrees C, respectively), suggesting that allosteric regulation of aconitase by this nucleotide may be involved in regulation of the tricarboxylic acid flux in oysters. Aconitase was less sensitive to ATP inhibition at 30 degrees C than at 20 degrees C, consistent with the elevated metabolic flux at higher temperatures. ADP and GDP also inhibited mitochondrial aconitase but at the levels well above the physiological concentrations of these nucleotides (6-11 mmol l(-1)). Our study shows expression of at least three UCP isoforms in C. virginica gill tissues but provides no indication that UCPs protect mitochondrial aconitase from oxidative inactivation in oysters. Overall, the results of this study indicate that temperature stress exaggerates toxicity of Cd leading to elevated oxidative stress in mitochondria, which may have important implications for survival of poikilotherms in polluted environments during seasonal warming and/or global climate change, and suggest a novel temperature-dependent mechanism of allosteric regulation of TCA flux in oyster mitochondria.
Aconitate Hydratase↗
Phe217 regulates the transfer of allosteric information across the subunit interface of the RecA protein filament.
BACKGROUND: ATP-mediated cooperative assembly of a RecA nucleoprotein filament activates the protein for catalysis of DNA strand exchange. RecA is a classic allosterically regulated enzyme in that ATP binding results in a dramatic increase in ssDNA binding affinity. This increase in ssDNA binding affinity results almost exclusively from an ATP-mediated increase in cooperative filament assembly rather than an increase in the inherent affinity of monomeric RecA for DNA. Therefore, certain residues at the subunit interface must play an important role in transmitting allosteric information across the filament structure of RecA. RESULTS: Using electron microscopic analysis of RecA polymer formation in the absence of DNA, we show that while wild-type RecA undergoes a slight decrease in filament length in the presence of ATP, a Phe217Tyr substitution results in a dramatic ATP-induced increase in cooperative filament assembly. Biosensor DNA binding measurements reveal that the Phe217Tyr mutation increases ATP-mediated cooperative interaction between RecA subunits by more than 250-fold. CONCLUSIONS: These studies represent the first identification of a subunit interface residue in RecA (Phe217) that plays a critical role in regulating the flow of ATP-mediated information throughout the protein filament structure. We propose a model by which conformational changes that occur upon ATP binding are propagated through the structure of a RecA monomer, resulting in the insertion of the Phe217 side chain into a pocket in the neighboring subunit. This event serves as a key step in intersubunit communication leading to ATP-mediated cooperative filament assembly and high affinity binding to ssDNA.
Adenosine Triphosphatases↗
Global conformational changes in allosteric proteins. A study of Escherichia coli cAMP receptor protein and muscle pyruvate kinase.
One of the basic features in allosteric regulation involves long range transduction of information. Based on crystallographic data on protein systems that are regulated by allosteric mechanisms, a global conformational change has always been observed. It is, therefore, important and useful to correlate the cooperativity of global structural change with the mode of binding of the regulatory ligand. Two systems were chosen for study, namely Escherichia coli cAMP receptor protein and muscle pyruvate kinase, which show negative and positive cooperativity in the binding of allosteric ligands, respectively. Quantitative titration of the global structural change, monitored by a high precision analytical gel chromatography technique, was conducted as a function of allosteric effector concentration. The results obtained for cAMP receptor protein show that the protein undergoes contraction upon binding of cAMP. The decreases in Stokes radius associated with complex formation are 0.1 +/- 0.1 and 0.7 +/- 0.1 A when one and two cAMP-binding sites are filled, respectively. The results for the pyruvate kinase system show a concerted structural change that quantitatively match the predicted behavior based on equilibrium constants derived from the analysis of steady state kinetic data by a two-state model. Hence, for these two systems, these results show that negative and positive cooperativity are correlated with sequential and concerted modes of structural change, respectively.
Allosteric Regulation↗
Cardiac sodium-calcium exchanger is regulated by allosteric calcium and exchanger inhibitory peptide at distinct sites.
The sarcolemmal Na+-Ca2+ exchanger (NCX) is the main Ca2+ extrusion mechanism in cardiac myocytes and is thus essential for the regulation of Ca2+ homeostasis and contractile function. A cytosolic region (f-loop) of the protein mediates regulation of NCX function by intracellular factors including inhibition by exchanger inhibitory peptide (XIP), a 20 amino acid peptide matching the sequence of an autoinhibitory region involved in allosteric regulation of NCX by intracellular Na+, Ca2+, and phosphatidylinositol-4,5-biphosphate (PIP2). Previous evidence indicates that the XIP interaction domain can be eliminated by large deletions of the f-loop that also remove activation of NCX by intracellular Ca2+. By whole-cell voltage clamping experiments, we demonstrate that deletion of residues 562-679, but not 440- 456, 498-510, or 680-685 of the f-loop selectively eliminates XIP-mediated inhibition of NCX expressed either heterologously (HEK293 and A549 cells) or in guinea pig cardiac myocytes. In contrast, by plotting I(NCX) against reverse-mode NCX-mediated Ca2+ transients in myocytes, we demonstrate that Ca2+-dependent regulation of NCX is preserved in Delta562-679, but significantly reduced in the other three deletion mutants. The findings indicate that f-loop residues 562-679 may contain the regulatory site for endogenous XIP, but this site is distinct from the Ca2+-regulatory domains of the NCX. Because regulation of the NCX by Na+ and PIP2 involves the endogenous XIP region, the Delta562-679 mutant NCX may be a useful tool to investigate this regulation in the context of the whole cardiac myocyte.
Action Potentials↗
Chronic agonist exposure induces down-regulation and allosteric uncoupling of the gamma-aminobutyric acid/benzodiazepine receptor complex.
Using [3H]flunitrazepam as a probe for the benzodiazepine-sensitive modulator site located on the gamma-aminobutyric acid (GABA)A receptor complex, we have investigated the cellular regulation of the GABAA receptor in neuronal cultures derived from embryonic chick brain. Treatment of cultures with 1 mM GABA for 48 hr causes a reversible 35% decrease in the number of [3H]flunitrazepam binding sites with no change in affinity. The EC50 for chronic GABA-induced down-regulation is 94 microM and the half-time is 25 hr. The effect of GABA is blocked by SR-95531, a GABAA receptor antagonist, and mimicked by muscimol but not baclofen. Consistent with the decrease in [3H]flunitrazepam binding, chronic GABA exposure causes a 43% decrease in the binding of [35S]t-butylbicyclophosphorothionate, a ligand for the receptor-associated chloride ionophore. In addition to chronic GABA-induced down-regulation, allosteric interactions between GABA and benzodiazepine recognition sites are uncoupled by 34%. The half-time and pharmacology for chronic GABA-induced uncoupling is indistinguishable from that for GABA-induced down-regulation, consistent with the hypothesis that the action of GABA at a common site induces both down-regulation and uncoupling.
Allosteric Regulation↗
Regulation of allosteric coupling and function of stably expressed gamma-aminobutyric acid (GABA)A receptors by chronic treatment with GABAA and benzodiazepine agonists.
Chronic treatments with drugs that stimulate or potentiate gamma-aminobutyric acid (GABA)A receptor function cause uncoupling of allosteric sites and downregulation of the GABAA receptors expressed in neurons. To study these effects on receptors having a defined subunit composition, we treated stably transfected mouse Ltk- cells (PA3 cells) with drugs known to uncouple GABAA receptors. Because dexamethasone controls the expression of bovine alpha-1, beta-1 and gamma 2L GABAA receptor subunit genes in PA3 cells, this expression system provides a way to study receptors in the absence of neuronal subunit gene promoters. We assayed binding of [3H]flunitrazepam to measure allosteric coupling and uptake of 36Cl- to measure GABAA receptor function. Chronic (4 day) treatment of PA3 cells with muscimol, GABA or flunitrazepam reduced the GABA enhancement of binding of [3H]flunitrazepam to PA3 cells. Chronic flurazepam or muscimol treatments also caused downregulation of benzodiazepine potentiation of muscimol-stimulated 36Cl- uptake. Chronic treatment with muscimol did not affect levels of subunit mRNAS and alpha 1- or beta 1-subunit protein of GABAA receptors and chronic flunitrazepam did not affect subunit mRNAs or alpha 1 protein. We conclude that chronic drug treatments regulate allosteric coupling and function of GABAA receptors in stably transfected cells and this regulation cannot be attributed to decreases in the expression of receptor subunits or to expression of subunits other than alpha 1 beta 1 or gamma 2L.
Allosteric Regulation↗
Target size analysis of skeletal muscle Ca2+ channels. Positive allosteric heterotropic regulation by d-cis-diltiazem is associated with apparent channel oligomer dissociation.
[3H]PN 200-110, a potent chiral benzoxadiazol 1,4-dihydropyridine Ca2+ antagonist was used to label guinea pig skeletal muscle Ca2+ channels. [3H]PN 200-110 binds with a Kd of approximately 1 nM to a homogeneous population of non-interacting binding sites; d-cis-diltiazem, but not l-cis-diltiazem increases the Bmax of [3H]PN 200-110 by 25% and slows the dissociation rate 3-fold at 37 degrees C. Target size analysis of the [3H]PN 200-110-labelled Ca2+ channels with 10 MeV electrons gave an Mr of 136 000 which was reduced to 75 000 by d-cis-diltiazem treatment of membranes. It is concluded that positive heterotropic allosteric regulation by d-cis-diltiazem is accompanied by channel oligomer dissociation.
Allosteric Regulation↗
Mechanisms of metalloregulation of an anion-translocating ATPase.
The ars (arsenical resistance) operon cloned from R-factor R773 has five genes that encode two repressor proteins, ArsR and ArsD, and three structural proteins, ArsA, ArsB, and ArsC. The ArsA and ArsB proteins form a membrane-bound pump that functions as an oxyanion-translocating ATPase. The substrates of the pump are the oxyanions arsenite or antimonite. The ArsC protein is an arsenate reductase that reduces arsenate to arsenite, which is subsequently pumped out of the cell. This review deals with the mechanism of transcriptional regulation by the ArsR repressor and allosteric regulation of the ArsA protein, the catalytic subunit of the pump. The chemical nature of the inducer plays an important role in regulation. In solution arsenite or antimonite exist as oxyanions and reacts with the cysteines in proteins. In both transcriptional regulation by the ArsR repressor and allosteric regulation of the ArsA ATPase, the ability of As(III) and Sb(III) to interact with the cysteines of the proteins, involves their action as effector.
Adenosine Triphosphatases↗
Supramolecular organization of glycolytic enzymes.
On the basis of the analysis of the data on adsorption of glycolytic enzymes to structural proteins of skeletal muscles and to the erythrocyte membranes, the data on enzyme-enzyme interactions and the data on the regulation of activity of glycolytic enzymes by cellular metabolites, the structure of the glycolytic enzymes complex adsorbed to a biological support has been proposed. The key role in the formation of multienzyme complex belongs to 6-phosphofructokinase. The enzyme molecule has two association sites, one of which provides the fixation of 6-phosphofructokinase on the support and another is saturated by fructose-1,6-bisphosphate aldolase. The multienzyme complex contains one tetrameric molecule of 6-phosphofructokinase and two molecules of each of other glycolytic enzymes. Hexokinase is not a part of the complex. The molecular mass of the multienzyme complex is about 2.6 X 10(6) daltons. The multienzyme complex has symmetry axis of second order. The formation of the multienzyme complex leads to the compartmentation of glycolytic process. The problem of integration of physico-chemical mechanisms of enzyme activity regulation (allosteric, dissociative and adsorptive mechanisms) is discussed.
Allosteric Regulation↗
Self-organizing molecular networks.
Strong diffusional mixing and short delivery times typical for micrometer and sub-micrometer reaction volumes lead to a special situation where the turnover times of individual enzyme molecules become the largest characteristic time scale of the chemical kinetics. Under these conditions, populations of cross-regulating allosteric enzymes form molecular networks that exhibit various kinds of self-organized coherent collective dynamics.
Allosteric Regulation↗
Determinants of specificity in coagulation proteases.
Proteases play diverse roles in a variety of essential biological processes, both as non-specific catalysts of protein degradation and as highly specific agents that control physiologic events. Here, we review the mechanisms of substrate specificity employed by serine proteases and focus our discussion on coagulation proteases. We dissect the interplay between active site and exosite specificity and how substrate recognition is regulated allosterically by Na+ binding. We also draw attention to a functional polarity that exists in the serine protease fold, which sheds light on the structural linkages between the active site and exosites.
Allosteric Regulation↗
Effects of 3-phosphoglycerate and other metabolites on the activation of AMP-activated protein kinase by LKB1-STRAD-MO25.
Skeletal muscle contraction results in the phosphorylation and activation of the AMP-activated protein kinase (AMPK) by an upstream kinase (AMPKK). The LKB1-STE-related adaptor (STRAD)-mouse protein 25 (MO25) complex is the major AMPKK in skeletal muscle; however, LKB1-STRAD-MO25 activity is not increased by muscle contraction. This relationship suggests that phosphorylation of AMPK by LKB1-STRAD-MO25 during skeletal muscle contraction may be regulated by allosteric mechanisms. In this study, we tested an array of metabolites including, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, 3-phosphoglycerate (3-PG), glucose 1-phosphate, glucose 1,6-bisphosphate, ADP, carnitine, acetylcarnitine, IMP, inosine, and ammonia for allosteric regulation. ADP inhibited both AMPK and LKB1-STRAD-MO25 actions, but probably is not important physiologically because of the low free ADP inside the muscle fiber. We found that 3-PG stimulated LKB1-STRAD-MO25 activity and allowed for increased AMPK phosphorylation. 3-PG did not stimulate LKB1-STRAD-MO25 activity toward the peptide substrate LKB1tide. These results have identified 3-PG as an AMPK-specific regulator of AMPK phosphorylation and activation by LKB1-STRAD-MO25.
AMP-Activated Protein Kinase Kinases↗
Oligomerization is a general mechanism for the activation of apoptosis initiator and inflammatory procaspases.
Proteolytic activation of initiator procaspases is a crucial step in the cellular commitment to apoptosis. Alternative models have been postulated for the activation mechanism, namely the oligomerization or induced proximity model and the allosteric regulation model. While the former holds that procaspases become activated upon proper oligomerization by an adaptor protein, the latter states that the adaptor is an allosteric regulator for procaspases. The allosteric regulation model has been applied for the activation of procaspase-9 by apoptotic protease-activating factor (Apaf-1) in an oligomeric complex known as the apoptosome. Using approaches that allow for controlled oligomerization, we show here that aggregation of multiple procaspase-9 molecules can induce their activation independent of the apoptosome. Oligomerization-induced procaspase-9 activation, both within the apoptosome and in artificial systems, requires stable homophilic association of the protease domains, raising the possibility that the function of Apaf-1 is not only to oligomerize procaspase-9 but also to maintain the interaction of the caspase-9 protease domain after processing. In addition, we provide biochemical evidence that other apoptosis initiator caspases (caspase-2 and -10) as well as a procaspase involved in inflammation (murine caspase-11) are also activated by oligomerization. Thus, oligomerization of precursor molecules appears to be a general mechanism for the activation of both apoptosis initiator and inflammatory procaspases.
Animals↗
Structure of Escherichia coli UMP kinase differs from that of other nucleoside monophosphate kinases and sheds new light on enzyme regulation.
Bacterial UMP kinases are essential enzymes involved in the multistep synthesis of nucleoside triphosphates. They are hexamers regulated by the allosteric activator GTP and inhibited by UTP. We solved the crystal structure of Escherichia coli UMP kinase bound to the UMP substrate (2.3 A resolution), the UDP product (2.6 A), or UTP (2.45 A). The monomer fold, unrelated to that of other nucleoside monophosphate kinases, belongs to the carbamate kinase-like superfamily. However, the phosphate acceptor binding cleft and subunit assembly are characteristic of UMP kinase. Interactions with UMP explain the high specificity for this natural substrate. UTP, previously described as an allosteric inhibitor, was unexpectedly found in the phosphate acceptor site, suggesting that it acts as a competitive inhibitor. Site-directed mutagenesis of residues Thr-138 and Asn-140, involved in both uracil recognition and active site interaction within the hexamer, decreased the activation by GTP and inhibition by UTP. These experiments suggest a cross-talk mechanism between enzyme subunits involved in cooperative binding at the phosphate acceptor site and in allosteric regulation by GTP. As bacterial UMP kinases have no counterpart in eukaryotes, the information provided here could help the design of new antibiotics.
Allosteric Regulation↗
The amino terminus regulates binding to and activation of cGMP-dependent protein kinase.
The allosteric regulation of binding to and the activation of cGMP-dependent protein kinase (cGMP kinase) was studied under identical conditions at 30 degrees C using three forms of cGMP-kinase which differed in the amino-terminal segment, e.g. native cGMP kinase, phosphorylated cGMP kinase which contained 1.4 +/- 0.4 mol phosphate/subunit and constitutively active cGMP kinase which lacked the amino-terminal dimerization domain. These three enzyme forms have identical kinetic constants, e.g. number of cGMP-binding sites, Km values for MgATP and the heptapeptide kemptide, and Vmax values. In the native enzyme, MgATP decreases the affinity for binding site 1. This effect is abolished by 1 M NaCl. In contrast, high concentrations of Kemptide increase the affinity of binding site 2 about fivefold. Under the latter conditions, identical Kd values of 0.2 microM were obtained for sites 1 and 2. Salt, MgATP and Kemptide do not affect the binding kinetics of the phosphorylated or the constitutively active enzyme, suggesting that allosteric regulation depends solely on the presence of a native amino-terminal segment. Cyclic GMP activates the native enzyme at Ka values which are identical with the Kd values for both binding sites. The activation of cGMP-dependent protein kinase is noncooperative but the Ka value depends on the substrate peptide concentration. These results show that the activity of cGMP kinase is primarily regulated by conformational changes within the amino-terminal domain.
Adenosine Triphosphate↗
Reversible high hydrostatic pressure inactivation of phosphofructokinase from Escherichia coli.
Tetrameric Escherichia coli phosphofructokinase dissociates reversibly on incubation under hydrostatic pressures of 80 MPa and above, yielding inactive dimers and monomers. The transition is dependent upon enzyme concentration and presence of ligands. The substrate, D-fructose 6-phosphate, which bridges the intersubunit interface at the active site, produces a massive stabilization to pressure, whereas ATP, which binds to only one subunit, induces only a mild stabilization. Both the positive allosteric regulator, GDP, and the negative allosteric regulator, phosphoenolpyruvate, whose binding sites lie at the other subunit interface, produce an intermediate effect. Of these ligands, only ATP increases the rate of reactivation after depressurization.
Adenosine Triphosphate↗
Multiple molecular determinants for allosteric modulation of alternatively spliced AMPA receptors.
Positive allosteric regulation of glutamate AMPA receptors involves conformational changes that can attenuate receptor desensitization and enhance ion flux through the channel pore. Many allosteric modulators (e.g., cyclothiazide and aniracetam) preferentially affect the flip (i) or flop (o) alternatively spliced isoform of AMPA receptors, implicating residues in the flip-flop domain as critical determinants of splice variant sensitivity. Indeed, previous mutational analyses have demonstrated that the differential sensitivity to cyclothiazide and aniracetam depends on a single amino acid, Ser (flip) and Asn (flop), suggesting that this residue may be solely responsible for differences in modulation of AMPA receptor isoforms. The present studies tested this hypothesis by investigating the molecular determinants of modulation of AMPA receptor splice variants by a structurally distinct compound, LY404187, which displays strikingly different and opposing kinetics of allosteric regulation characterized by a time-dependent enhancement in potentiation of homomeric GluR1-GluR4i and a time-dependent reduction in potentiation of GluR1-GluR4o. Site-directed mutagenesis of residues in the flip-flop domain of GluR2 revealed that, although exchange of Asn775 for Ser in GluR2o was sufficient to confer the GluR2i phenotype of potentiation, the corresponding mutation, Ser775Asn, in GluR2i did not impart the GluR2o response. In fact, the GluR2o kinetics of modulation depended on a novel set of substitutions in GluR2i, including Thr765Asn, Pro766Ala, and Val779Leu in combination with Ser775Asn. Collectively, these results show that, unlike cyclothiazide and aniracetam, the residues that confer splice variant differences in modulation by LY404187 are not identical and indicate that allosteric regulation of AMPA receptors can arise from multiple molecular determinants.
Allosteric Regulation↗