ABSTRACT
Pyruvate kinase isoform M2 (PKM2) converts phosphoenolpyruvate (PEP) to pyruvate and plays an important role in cancer metabolism. Here, we show that post-translational modifications and a patient-derived mutation regulate pyruvate kinase activity of PKM2 through modulating the conformation of the PKM2 tetramer. We determined crystal structures of human PKM2 mutants and proposed a "seesaw" model to illustrate conformational changes between an inactive T-state and an active R-state tetramers of PKM2. Biochemical and structural analyses demonstrate that PKM2(Y105E) (phosphorylation mimic of Y105) decreases pyruvate kinase activity by inhibiting FBP (fructose 1,6-bisphosphate)-induced R-state formation, and PKM2(K305Q) (acetylation mimic of K305) abolishes the activity by hindering tetramer formation. K422R, a patient-derived mutation of PKM2, favors a stable, inactive T-state tetramer because of strong intermolecular interactions. Our study reveals the mechanism for dynamic regulation of PKM2 by post-translational modifications and a patient-derived mutation and provides a structural basis for further investigation of other modifications and mutations of PKM2 yet to be discovered.
Subject(s)
Humans , Acetylation , Allosteric Regulation , Carrier Proteins , Chemistry , Genetics , Metabolism , Crystallography, X-Ray , Fructosediphosphates , Chemistry , Metabolism , Gene Expression , Kinetics , Membrane Proteins , Chemistry , Genetics , Metabolism , Models, Molecular , Mutation , Neoplasms , Genetics , Pathology , Phosphorylation , Protein Conformation , Protein Multimerization , Protein Processing, Post-Translational , Protein Subunits , Chemistry , Genetics , Metabolism , Thyroid Hormones , Chemistry , Genetics , Metabolism , Tumor Cells, CulturedABSTRACT
Background BmK I, a site-3-specific modulator of voltage-gated sodium channels (VGSCs), causes pain and hyperalgesia in rats, while BmK IT2, a site-4-specific modulator of VGSCs, suppresses pain-related responses. A stronger pain-related effect has been previously attributed to Buthus martensi Karsch (BmK) venom, which points out the joint pharmacological effect in the crude venom.Methods In order to detect the joint effect of BmK I and BmK IT2 on ND7-23 cells, the membrane current was measured by whole cell recording. BmK I and BmK IT2 were applied successively and jointly, and the synergistic modulations of VGSCs on ND7-23 cells were detected.Results Larger peak I Na and more negative half-activation voltage were elicited by joint application of BmK I and BmK IT2 than by application of BmK I or BmK IT2 alone. Compared to the control, co-applied BmK I and BmK IT2 also significantly prolonged the time constant of inactivation.Conclusions Our results indicated that site-4 toxin (BmK IT2) could enhance the pharmacological effect induced by site-3 toxin (BmK I), suggesting a stronger effect elicited by both toxins that alone usually exhibit opposite pharmacological effects, which is related to the allosteric interaction between receptor site 3 and site 4. Meanwhile, these results may bring a novel perspective for exploring the underlying mechanisms of scorpion sting-induced pain.(AU)
Subject(s)
Animals , Allosteric Regulation , Scorpion Stings , HyperalgesiaABSTRACT
Insulin is a hormone that is essential for regulating energy storage and glucose metabolism in the body. Insulin in liver, muscle, and fat tissues stimulates the cell to take up glucose from blood and store it as glycogen in liver and muscle. Failure of insulin control causes diabetes mellitus (DM). Insulin is the unique medicine to treat some forms of DM. The population of diabetics has dramatically increased over the past two decades, due to high absorption of carbohydrates (or fats and proteins), lack of physical exercise, and development of new diagnostic techniques. At present, the two largest developing countries (India and China) and the largest developed country (United States) represent the top three countries in terms of diabetic population. Insulin is a small protein, but contains almost all structural features typical of proteins: α-helix, β-sheet, β-turn, high order assembly, allosteric T®R-transition, and conformational changes in amyloidal fibrillation. More than ten years' efforts on studying insulin disulfide intermediates by NMR have enabled us to decipher the whole picture of insulin folding coupled to disulfide pairing, especially at the initial stage that forms the nascent peptide. Two structural switches are also known to regulate insulin binding to receptors and progress has been made to identify the residues involved in binding. However, resolving the complex structure of insulin and its receptor remains a challenge in insulin research. Nevertheless, the accumulated knowledge of insulin structure has allowed us to specifically design a new ultra-stable and active single-chain insulin analog (SCI-57), and provides a novel way to design super-stable, fast-acting and cheaper insulin formulations for DM patients. Continuing this long journey of insulin study will benefit basic research in proteins and in pharmaceutical therapy.
Subject(s)
Animals , Humans , Allosteric Regulation , Amino Acid Sequence , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Insulin , Chemistry , Genetics , Metabolism , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Engineering , Protein Folding , Protein Stability , Protein Structure, Tertiary , Receptor, Insulin , Chemistry , Metabolism , Surface PropertiesABSTRACT
A oxigenação da Hb humana (HbA) requer um modelo detrês estado: dois estados desoxi To e Tx, livre e complexado comanions, respectivamente e um estado R oxi. A regulação entre essesestados é modulada pela presença de anions, tais como cloreto,que se ligam ao estado T. A ligação de cloreto, entretanto, permanececontroversa. O objetivo deste trabalho foi o estudo dasargininas 92α (interface α1β2) e 141α (C-terminal) como sítios deligação de cloreto. Para isso, estudamos Hbs com mutações sitiodirigidas: mutantes naturais Hb J Cape Town (R92Q), desArg(R141Δ), Chesapeake (R92L) e a construída Chesapeake desArg(R92L,141Δ). As Hbs foram expressas em Escherichia coli epurificadas. Através de curvas de oxigenação, medimos afinidade ecooperatividade, variando a atividade de água e efeito Bohr napresença e ausência de cloreto. Características estruturais foramobtidas por espectroscopia 1H RMN. Parâmetros de oxigenação eEfeito Bohr medidos indicaram uma afinidade maior e cooperatividademenor na ausência e presença de cloreto para todosos mutantes. Modificações estruturais representaram aspectosfuncionais das Hbs modificadas, como aumento significativo naafinidade e mudanças na cooperatividade. Estudos da atividade deágua em função da concentração de cloreto revelaram que a HbdesArg foi a única Hb modificada que seguiu o modelo de trêsestados. As demais não apresentaram o estado Tx, fato confirmadopelo número de moléculas de água ligadas durante a transiçãodesoxi-oxi. Este comportamento sugere que a Arginina 92 poderiaser responsável pela ligação de cloreto, uma vez que a oxigenaçãodas Hbs sem esse resíduo não pode ser ajustada pelo modelo detrês estados. Contudo, o efeito Bohr mostrou que todas as Hbsmodificadas liberaram ~ 1 próton na presença de cloreto, diferenteda HbA que libera ~ 2, sugerindo um papel para arginina 141 noefeito Bohr terciário e quaternário.
The oxygenation of human Hb (HbA) demands a three statemodel: two deoxy states To and Tx, free and complexed withanions respectively, and an oxy R state. The regulation betweenthese states is modulated by the presence of anions, such as chloride,that binds to T state. The binding of chloride, however, remainscontroversial. The aim of this work is the study of arginines 92a(a1ß2 interface) and 141a (C-terminal ) as chloride binding sites.To investigate that, we have studied 92 and 141 site directedmutant species: natural mutants Hb J-Cape-Town (R92Q), desArg(R141Δ), Chesapeake (R92L), and the constructed ChesapeakedesArg (R92L,141Δ). We expressed Hbs in Escherichia coli andpurified. Through oxygen binding curves we measured affinityand cooperativity, in function of water effect and Bohr effect inpresence and absence of chloride. Structural features were obtainedthrough 1H NMR spectroscopy Oxygen binding properties andBohr effect measured indicated a higher affinity and lowercooperativity in absence and presence of chloride for all mutants.Structural changes represent functional aspects of mutant Hbs such as a significant rise in affinity or a change in cooperativity.Water activity studies conducted as a function of chlorideconcentration showed that the only Hb desArg follows the threestate model. The other mutant Hbs do not exhibit the Tx state, afact confirmed by the number of water molecules bound to eachHb during the deoxy-oxy transition. This behavior suggests thatthe Arginine 92 site could be responsible for chloride binding toHb, since oxygenation of 92 mutant Hbs cannot be adjusted bythe three state model. However, Bohr effect showed that all mutantHbs released~1 proton in chloride presence, different from HbAthat releases ~2, suggesting a role for 141 arginine in the tertiaryand quaternary Bohr effect.
Subject(s)
Humans , Allosteric Regulation , Binding Sites , Erythrocytes , OxygenationABSTRACT
Vertebrate hemoglobin, contained in erythrocytes, is a globular protein with a quaternary structure composed of 4 globin chains (2 alpha and 2 beta) and a prosthetic group named heme bound to each one. Having myoglobin as an ancestor, hemoglobin acquired the capacity to respond to chemical stimuli that modulate its function according to tissue requirements for oxygen. Fish are generally submitted to spatial and temporal O2 variations and have developed anatomical, physiological and biochemical strategies to adapt to the changing environmental gas availability. Structurally, most fish hemoglobins are tetrameric; however, those from some species such as lamprey and hagfish dissociate, being monomeric when oxygenated and oligomeric when deoxygenated. Fish blood frequently possesses several hemoglobins; the primary origin of this finding lies in the polymorphism that occurs in the globin loci, an aspect that may occasionally confer advantages to its carriers or even be a harmless evolutionary remnant. On the other hand, the functional properties exhibit different behaviors, ranging from a total absence of responses to allosteric regulation to drastic ones, such as the Root effect.
Subject(s)
Animals , Adaptation, Physiological , Fishes/physiology , Hemoglobins , Allosteric Regulation , Biological Evolution , Fishes/metabolism , Hemoglobins/chemistry , Hemoglobins/genetics , Hemoglobins/metabolismABSTRACT
6-phosphofructo-1-kinase (phosphofructokinase; PFK) activity from Rhodnius prolixus, a haematophagous insect which is usually a poor flyer, was measured and compared in two metabolically active tissues - flight muscle and fat body. The activity of this important regulatory glycolytic enzyme was much more pronounced in muscle (15.1 ± 1.4 U/mg) than in fat body extracts (3.6±0.4 U/mg), although the latter presented higher levels of enzyme per protein content, as measured by western-blotting. Muscle extracts are more responsible than fat body to ATP and fructose 6-phosphate, both substrates of PFK. Allosteric regulation exerted by different effectors such as ADP, AMP and fructose 2,6-phosphate presented a singular pattern for each tissue. Optimal pH (8.0-8.5) and sensitivity to pH variation was very similar, and citrate was unable to inhibit PFK activity in both extracts. Our results suggest the existence of a particular PFK activity for each tissue, with regulatory patterns that are consistent with their physiological roles.
A atividade da fosfofrutocinase (PFK) de Rodnius prolixus, um inseto hematófago, o qual vôa somente pequenas distâncias, foi medida e comparada em dois tecidos metabolicamente ativos - músculo de asa e corpo gorduroso. A atividade desta importante enzima glicolítica regulatória foi muito mais pronunciada em músculo de asa (15,1 ±1,4 U/mg) do que em extrato de corpo gorduroso (3,6 ±0,4 U/mg) embora este último tenha apresentado níveis mais altos da enzima por quantidade de proteína, como medido por western-blotting. Extratos de músculo foram mais responsivos do que corpo gorduroso para ATP e frutose-6-fosfato, ambos substratos da PFK. A regulação alostérica exercida por diferentes efetores tais como ADP, AMP, frutose-2,6-bisfosfato apresentou um padrão singular para cada tecido. O pH ótimo (8,0-8,5) e a sensibilidade a variações de pH, foram muito similares e o citrato foi incapaz de inibir a atividade da PFK em ambos os extratos. Nossos resultados sugerem a existência de uma atividade particular da PFK para cada tecido com padrões regulatórios que são consistentes com suas funções fisiológicas.
Subject(s)
Animals , Fat Body/enzymology , Muscle, Skeletal/enzymology , Phosphofructokinase-1/metabolism , Phosphofructokinase-1/physiology , Rhodnius/enzymology , Allosteric Regulation/physiology , Blotting, Western , KineticsABSTRACT
Large conductance Ca2+-activated K+ (BK) channels belong to the S4 superfamily of K+ channels that include voltage-dependent K+ (Kv) channels characterized by having six (S1-S6) transmembrane domains and a positively charged S4 domain. As Kv channels, BK channels contain a S4 domain, but they have an extra (S0) transmembrane domain that leads to an external NH2-terminus. The BK channel is activated by internal Ca2+, and using chimeric channels and mutagenesis, three distinct Ca2+-dependent regulatory mechanisms with different divalent cation selectivity have been identified in its large COOH-terminus. Two of these putative Ca2+-binding domains activate the BK channel when cytoplasmic Ca2+ reaches micromolar concentrations, and a low Ca2+ affinity mechanism may be involved in the physiological regulation by Mg2+. The presence in the BK channel of multiple Ca2+-binding sites explains the huge Ca2+ concentration range (0.1 ìM-100 ìM) in which the divalent cation influences channel gating. BK channels are also voltage-dependent, and all the experimental evidence points toward the S4 domain as the domain in charge of sensing the voltage. Calcium can open BK channels when all the voltage sensors are in their resting configuration, and voltage is able to activate channels in the complete absence of Ca2+. Therefore, Ca2+ and voltage act independently to enhance channel opening, and this behavior can be explained using a two-tiered allosteric gating mechanism.
Subject(s)
Animals , Calcium Channels/physiology , Large-Conductance Calcium-Activated Potassium Channels/physiology , Allosteric Regulation/physiology , Ion Channel Gating/physiology , Membrane Potentials/physiologyABSTRACT
<p><b>BACKGROUND</b>Atrial fibrillation (AF) is the most common supraventricular arrhythmia in clinical practice. Chronic atrial fibrillation (CAF) is associated with ionic remodeling. However, little is known about the activity of ATP-sensitive potassium current (IK, ATP) during CAF. So we studied the changes of IK, ATP density and allosteric modulation of ATP-sensitivity by intracellular pH during CAF.</p><p><b>METHODS</b>Myocardium samples were obtained from the right auricular appendage of patients with rheumatic heart disease complicated with valvular disease in sinus rhythm (SR) or CAF. There were 14 patients in SR group and 9 patients in CAF group. Single atrial cells were isolated using an enzyme dispersion technique. IK, ATP was recorded using the whole-cell and inside-out configuration of voltage-clamp techniques. In whole-cell model, myocytes of SR and CAF groups were perfused with simulated ischemic solution to elicit IK, ATP. In inside-out configuration, the internal patch membranes were exposed to different ATP concentrations in pH 7.4 and 6.8.</p><p><b>RESULTS</b>Under simulated ischemia, IK, ATP current density of CAF group was significantly higher than in SR group [(83.5 +/- 10.8) vs. (58.7 +/- 8.4) pA/pF, P < 0.01]. IK, ATP of the two groups showed ATP concentration-dependent inhibition. The ATP concentration for 50% current inhibition (IC50) for the SR group was significantly different in pH 7.4 and pH 6.8 (24 vs. 74 micromol/L, P < 0.01). The IC50 did not change significantly in CAF group when the pH decreased from 7.4 to 6.8.</p><p><b>CONCLUSIONS</b>During CAF, IK, ATP current density was increased and its allosteric modulation of ATP-sensitivity by intracellular pH was diminished.</p>
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Adenosine Triphosphate , Pharmacology , Allosteric Regulation , Atrial Fibrillation , Metabolism , Chronic Disease , Dose-Response Relationship, Drug , Hydrogen-Ion Concentration , Potassium Channels , PhysiologyABSTRACT
O ácido metilpropiônico (RSR13) é um modificador alostérico da hemoglobina, com a qual se liga de forma não-covalente, diminuindo sua afinidade pelo oxigênio de modo dose-dependente e, conseqüentemente, aumentando a oxigenação periférica. O objetivo deste artigo é apresentar brevemente as evidências científicas acerca das características farmacológicas e funcionais, indicações médicas e efeitos adversos do uso do RSR13 pro atletas, a mais recente alternativa de aumento artificial do desempenho. Estudos experimentais preliminares verificaram algum efeito positivo do RSR13 sobre a recuperação do miocárdio isquêmico e sobre a extensão da isquemia cerebral, mas as principais indicações estudadas atualmente são a cirurgia com hipotermia e cardioplegia durante circulação extracorpórea e o uso como agente coadjuvante potenciador da radioterapia para certos tumores sólidos. Somente um estudo em modelo canino mostrou aumento do consumo máximo de oxigênio em músculo isolado, não existindo evidências de que o RSR13 possa efetivamente melhorar o desempenho em humanos. Em realidade, já foram descritos efeitos adversos, como diminuição da perfusão sanguínea, elevação da pressão arterial e diminuição da função renal. Antecipando o potencial aumento da utilização do RSR13 por atletas, métodos já foram desenvolvidos para sua detecção em amostras de urina humana
Subject(s)
Humans , Propionates/adverse effects , Oxygen Consumption , Muscle Contraction , Doping in Sports , Hemoglobin A , Oxygen , Allosteric Regulation , Sports , Substance Abuse DetectionABSTRACT
Carbamoyl phosphate synthetase (CPS) activity in Streptomyces lividans was repressed (70%) by addition of arginine and uracil in the growth medium. Enzyme activity was also inhibited by UMP and activated by ornithine and IMP. Pattern of inhibition and activation was similar irrespective of whether the cells were grown in medium supplemented with arginine or with uracil. A mutant of S. coelicolor with dual auxotrophy for arginine and uracil possessed only about 20% of CPS activity compared to the wild-type strain. An activity staining protocol has been developed for CPS enzyme. Using this method a single CPS band has been observed in the crude extracts of Escherichia coli as well as in S. lividans. Taken together, our results supported the conclusion that Streptomyces species might possess a single CPS enzyme unlike other gram-positive bacteria, which show the presence of two pathway-specific isozymes (Bacillus) or none (Lactobacillus and Leuconostoc).
Subject(s)
Allosteric Regulation , Arginine/pharmacology , Carbamyl Phosphate/metabolism , Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor/genetics , Colorimetry , Escherichia coli , Gene Expression Regulation, Bacterial , Glutamine/metabolism , Magnesium , Mutation , Radiometry , Streptomyces/drug effects , Uracil/pharmacologyABSTRACT
DNA binding compounds were previously shown to bind to the right-handed DNA forms and hybrid B-Z forms in a highly cooperative manner and indicate that structural specificity plays a key role in a ligand binding to DNA. In this study, the modes of binding and structural specificity of agents to unusual DNA are examined by a variety of fluorescence techniques (intensity, polarization and quenching, etc.) to explore a reliable method to detect the association environment of ligands to deoxyoligonucleotides initially containing a B-Z junction between the left-handed Z-DNA and right-handed B-DNA. The results of fluorescence energy transfer measurement demonstrated that the ligand molecules bind to the allosterically converted DNA structures by intercalation. In the absence of high-resolution structural data, this fluorescence energy transfer measurement allowed reliable measures and infer the binding environment of ligands to the allosteric DNA structures.
Subject(s)
Allosteric Regulation , Circular Dichroism , DNA/chemistry , Energy Transfer , Ethidium/metabolism , Exodeoxyribonucleases/metabolism , Ligands , Motion , Nucleic Acid Conformation , Oligodeoxyribonucleotides/metabolismABSTRACT
The specific association of drugs with deoxyoligonucleotides, containing a B-Z junction between left-handed Z-DNA and right-handed B-DNA, was examined by fluorescence and circular dichroism (CD) technique. Ethidium was chosen for a simple DNA binding compound because it binds to right-handed DNA and hybrid B-Z forms containing a B-Z junction in a highly cooperative manner. The binding isotherms were analyzed by an allosteric model in order to describe the cooperativity of association. Binding of ethidium to the DNA that are initially in the hybrid B-Z forms showed over an order of magnitude higher affinity than other DNA which were entirely in the B-form. The conformational transitions of deoxyoligonucleotides containing a B-Z junction as a result of ethidium binding were monitored by CD and the influence of NaCl on the complex formation was also determined by the CD spectra. The singular value decomposition (SVD) analysis was used to characterize a family of CD spectra of the species in binding equilibria. The results of SVD analysis showed a strikingly complex thermodynamic equilibria of cooperative binding of drugs to the allosterically converted DNA forms. The results also showed that these DNA forms in low- and high-salt were different in the absence or presence of drug. These results demonstrate that DNA-binding-drugs can preferentially interact with specific DNA structures and that these interactions are accompanied by allosteric changes of DNA conformations.
Subject(s)
Allosteric Regulation/genetics , Circular Dichroism , DNA/chemistry , Ethidium/chemistry , Fluorescent Dyes/chemistry , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemistry , Sodium Chloride/pharmacology , ThermodynamicsABSTRACT
The molecular basis underlying the anxiolytic, sedative hypnotic properties of the benzodiazepine compounds are examined at the ligh of recent progress in the field of psychopharmacology and molecular biology. The benzodiazepine receptors, originally characterized as the building site of radiolabelled benzodiazepines, are now identified as allosteric domains of a small population of the plethora of putative brain GABA-A receptor complexes. Emphasis is given to the understanding of the molecular biology of the GABA-A receptor complex and the hypothesis of the multiplicity of benzodiazepine receptors. Experiments describing the need of the gamma subunit of the GABA-A receptor for full benzodiazepine action as positive or negative allosteric modulators of the GABA-A receptor complez are analyzed. Likewise, experiments differentiating the barbiturate receptor from that of the benzodiazepines, alcohol and anesthetic agents are discussed ath the light of recent progress in the molecular biology of the GABA-A receptor. It is concluded that the GABA-A receptor complex is regulated at multiple sites, by a variety of drugs. Regarding putative endogenous ligands for the benzodiazepine receptor, evidences are discussed in support of the endozepines and their implications in health and diseases
Subject(s)
Humans , Benzodiazepines/pharmacology , Receptors, GABA-A/drug effects , Allosteric Regulation , Anxiety/drug therapyABSTRACT
Chemical modification is usually employed to study enzyme active sites. Valuable information can also be obtained, however, when this technique is used to probe allosteric sites. This approach is discussed in this article, and it is exemplified in chemical modification studies of the allosteric enzyme phosphofructokinase
Subject(s)
Allosteric Regulation/physiology , Allosteric Site/physiology , Enzyme Activation/physiology , Ligands , Mutagenesis, Site-Directed/physiology , Phosphofructokinase-1/chemistry , Protein Conformation , Sulfhydryl Compounds/chemistry , Adenosine Triphosphate/chemistry , Citrates/chemistry , Fructose/chemistryABSTRACT
Transcription is the foremost event in gene expression in which the enzyme RNA polymerase copies the genetic information from DNA to RNA. Much of our understanding of this process have come from studies carried out in Escherichia coli. A faithful and efficient transcription machinery of E. coli can be reconstituted in vitro with purified RNA polymerase and promoter-containing DNA. It is generally believed that in E. coli and most other organisms, the control of gene expression lies with the initiation of transcription. In this review, an attempt has been made to understand the mechanistic details of the initiation of transcription from the structural point of view of the promoter and the RNA polymerase. Allosteric nature of the enzyme has also been discussed at the end.
Subject(s)
Allosteric Regulation , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/genetics , Models, Genetic , Promoter Regions, Genetic , Transcription, GeneticABSTRACT
Amiloride (AM) is a well known potassium sparing diuretic. The effects of AM at the cellular level include blockade of Na+/H+ exchange in several tissues and inhibition of passive sodium flux in epithelial cells. In this study we have explored the interactions of amiloride with muscarinic receptors, using isolated rat tracheal rings and compared its effects to those of the muscarinic receptor subtype-selective antagonist pirenzepine (PZ). The results obtained demonstrate the ability of AM (100 uM to 1mM) to inhibit the ACh induced rat tracheall contractions. The inhibition resulted in the reduction of the Emax values of ACh in this preparation, and the apparent Ki for AM was of 478 uM. This effect was also observed in a sodium-free choline medium, indicating that it is independent from sodium transport mechanisms sensitive to AM. In contrast to AM, PZ displayed a surmountable type of antagonism with a pA2 value of 6.52. The results demonstrate a differential antagonism by AM and PZ of the muscarinic receptors present in the smooth muscle of the rat trachea