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At least 721 records · Page 40Linked to original sources
PubMed2001

Role of two different glyceraldehyde-3-phosphate dehydrogenases in controlling the reversible Embden-Meyerhof-Parnas pathway in Thermoproteus tenax: regulation on protein and transcript level.

The hyperthermophilic archaeum Thermoproteus tenax uses a variant of the Embden-Meyerhof-Parnas (EMP) pathway as the main route for carbohydrate metabolism. This variant is characterized by a reversible nonallosteric PPi-dependent phosphofructokinase and two glyceraldehyde-3-phosphate dehydrogenases differing in cosubstrate specificity, phosphate dependence, and allosteric behavior. Although the nonphosphorylating NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN; E.C. 1.2.1.8) fulfills exclusively catabolic purposes, the phosphorylating NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (NADP+-GAPDH; E.C. 1.2.1.13) exhibits anabolic features. The gene encoding the NADP+-GAPDH was cloned, sequenced, and expressed in Escherichia coli. The deduced protein sequence displayed 47%-53% sequence identity to archaeal phosphorylating GAPDHs. The kinetic parameters of the NADP+-GAPDH showed a clear preference for the reductive reaction with a 5-fold-higher specific activity in the reductive reaction as compared to the oxidative reaction and a 20-fold-lower Km for 1,3-bisphosphoglycerate as compared to glyceraldehyde-3-phosphate. Contrary to GAPN, the enzyme is not allosterically regulated. The coding gene overlaps by 1 bp with a preceding open reading frame coding for 3-phosphoglycerate kinase (PGK; E.C. 2.7.2.3). Northern analyses identified mono- and bicistronic messages of both genes in an equimolar ratio. Transcript levels and specific activity of NADP+-GAPDH and PGK were 3- to 4-fold higher under autotrophic conditions as compared to heterotrophic conditions, whereas transcript abundance and specific activity of GAPN remained constant in autotrophically and heterotrophically grown cells. The different regulation of the two counteracting glyceraldehyde-3-phosphate dehydrogenases is discussed with respect to the flux control of the T. tenax-specific EMP variant.

Amino Acid Sequence↗
PubMed2005

Regulation of protein kinase C delta by phorbol ester, endothelin-1, and platelet-derived growth factor in cardiac myocytes.

Protein kinase C (PKC) delta is regulated allosterically by phosphatidylserine and diacylglycerol (which promote its translocation to the membrane) and by phosphorylation of Ser/Thr and Tyr residues. Although phosphorylation on Thr-505/Ser-643/Ser-662 may simply "prime" PKCdelta for activation, it could be regulatory. We examined the regulation of PKCdelta in cardiac myocytes by endothelin-1 (Gq protein-coupled receptor agonist) and platelet-derived growth factor (receptor tyrosine kinase agonist) in comparison with phorbol 12-myristate 13-acetate (PMA). All increased phosphorylation of PKCdelta(Thr-505/Ser-643) and of Tyr residues, although to differing extents. De novo phosphorylation occurred mainly after translocation of PKCdelta to the particulate fraction, and phosphorylations of Thr-505/Ser-643 versus Tyr residues were essentially independent events. Following chromatographic separation of the PKCdelta subspecies, activities were correlated with immunoreactivity profiles of total and phosphorylated forms. In unstimulated cells, approximately 25% of PKCdelta lacked phosphorylation of Thr-505/Ser-643 and displayed minimal activity (assayed in the presence of phosphatidylserine/PMA following chromatography). Endothelin-1 or PMA (10 min) promoted Thr-505/Ser-643 phosphorylation of this pool, and this was associated with an increase in total recoverable PKCdelta activity. Meanwhile, in cells exposed to endothelin-1 or PMA, the overall pool of PKCdelta translocated rapidly (30 s) to the particulate fraction and was phosphorylated on Tyr residues. This was associated with an increase in lipid-independent activity (i.e. the phosphatidylserine/PMA requirement disappeared). For endothelin-1, Tyr phosphorylation of PKCdelta and the increase in phosphatidylserine/PMA-independent activity persisted after PKCdelta retrotranslocated to the soluble fraction. We concluded that, with this physiological agonist, PKCdelta becomes activated in the particulate fraction but retains activity following its retrotranslocation, presumably to phosphorylate substrates elsewhere.

Animals↗
PubMed2004

Crystal structure of circadian clock protein KaiA from Synechococcus elongatus.

The circadian clock found in Synechococcus elongatus, the most ancient circadian clock, is regulated by the interaction of three proteins, KaiA, KaiB, and KaiC. While the precise function of these proteins remains unclear, KaiA has been shown to be a positive regulator of the expression of KaiB and KaiC. The 2.0-A structure of KaiA of S. elongatus reported here shows that the protein is composed of two independently folded domains connected by a linker. The NH(2)-terminal pseudo-receiver domain has a similar fold with that of bacterial response regulators, whereas the COOH-terminal four-helix bundle domain is novel and forms the interface of the 2-fold-related homodimer. The COOH-terminal four-helix bundle domain has been shown to contain the KaiC binding site. The structure suggests that the KaiB binding site is covered in the dimer interface of the KaiA "closed" conformation, observed in the crystal structure, which suggests an allosteric regulation mechanism.

Allosteric Site↗
PubMed1994

Isolation and characterization of a novel acetyl-CoA carboxylase kinase from rat liver.

Acetyl-CoA carboxylase is regulated allosterically by citrate and covalently by a phosphorylation/dephosphorylation mechanism. We have isolated and purified from rat livers a novel kinase that phosphorylates and inactivates the carboxylase. This kinase is bound to the carboxylase and can be eluted in salt-rich solution. The native kinase exists as high molecular weight aggregates of a subunit that has a molecular weight of 40,000. The phosphorylation sites of the carboxylase were determined after tryptic and cyanogen bromide digestions of 32P-labeled carboxylase and separation of the peptides by various chromatographic procedures. Amino acid analyses of the phosphopeptides showed that the Ser77 and Ser1200 residues were the sites of phosphorylation. Treating the phosphorylated carboxylase with the Mn(2+)-dependent acetyl-CoA carboxylase phosphatase 2 removed the phosphate and reactivated the carboxylase. These results suggest that both this kinase and the acetyl-CoA carboxylase phosphatase 2 act at the same site(s) in the acetyl-CoA carboxylase molecule. Citrate dramatically inhibits the kinase-mediated phosphorylation of the carboxylase, suggesting that the allosteric modification and activation by citrate render the phosphorylation sites inaccessible to the kinase and therefore maintain high carboxylase activity. This observation indicates that there is a close interplay between the citrate effect on and phosphorylation of the carboxylase in regulating its activity.

Acetyl-CoA Carboxylase↗
PubMed2003

Control of tetrapyrrole biosynthesis by alternate quaternary forms of porphobilinogen synthase.

Porphobilinogen synthase (PBGS) catalyzes the first common step in the biosynthesis of tetrapyrroles (such as heme and chlorophyll). Although the predominant oligomeric form of this enzyme, as inferred from many crystal structures, is that of a homo-octamer, a rare human PBGS allele, F12L, reveals the presence of a hexameric form. Rearrangement of an N-terminal arm is responsible for this oligomeric switch, which results in profound changes in kinetic behavior. The structural transition between octamer and hexamer must proceed through an unparalleled equilibrium containing two different dimer structures. The allosteric magnesium, present in most PBGS, has a binding site in the octamer but not in the hexamer. The unprecedented structural rearrangement reported here relates to the allosteric regulation of PBGS and suggests that alternative PBGS oligomers may function in a magnesium-dependent regulation of tetrapyrrole biosynthesis in plants and some bacteria.

Allosteric Site↗
PubMed1996

[Inducing conformational changes in thrombin by ligands as a way of regulating its activity].

This review summarizes data on changes in thrombin conformation induced by various ligands including hirudin, hirugen, similar acidic peptides, fibrinogen, thrombomodulin, heparin, antithrombin, prothrombin, and platelet receptor. Studies of enzyme kinetics, spectroscopy, X-ray structure, and thermodynamics of thrombin complexes with various ligands indicate that the thrombin molecule undergoes conformational changes. Possible mechanisms of allosteric regulation of thrombin activity by high molecular weight ligands are discussed which can be important during physiologic or pathophysiologic reactions of the organism.

Antithrombin III↗
PubMed2006

Physiological consequences of loss of allosteric activation of yeast NAD+-specific isocitrate dehydrogenase.

Based on allosteric regulatory properties, NAD+-specific isocitrate dehydrogenase (IDH) is believed to control flux through the tricarboxylic acid cycle in vivo. To distinguish growth phenotypes associated with regulatory dysfunction of this enzyme in Saccharomyces cerevisiae, we analyzed strains expressing well defined mutant forms of IDH or a non-allosteric bacterial NAD+-specific isocitrate dehydrogenase (IDHa). As previously reported, expression of mutant forms of IDH with severe catalytic defects but intact regulatory properties produced an inability to grow with acetate as the carbon source and a dramatic increase in the frequency of generation of petite colonies, phenotypes also exhibited by a strain (idh1Deltaidh2Delta) lacking IDH. Reduced growth rates on acetate medium were also observed with expression of enzymes with severe regulatory defects or of the bacterial IDHa enzyme, suggesting that allosteric regulation is also important for optimal growth on this carbon source. However, expression of IDHa produced no effect on petite frequency, suggesting that the intermediate petite frequencies observed for strains expressing regulatory mutant forms of IDH are likely to correlate with the slight reductions in catalytic efficiency observed for these enzymes. Finally, rates of increase in oxygen consumption were measured during culture shifts from medium with glucose to medium with ethanol as the carbon source. Strains expressing wild-type or catalytically deficient mutant forms of IDH exhibited rapid respiratory transitions, whereas strains expressing regulatory mutant forms of IDH or the bacterial IDHa enzyme exhibited much slower respiratory transitions. This suggests an important physiological role for allosteric activation of IDH during changes in environmental conditions.

Allosteric Site↗
PubMed2006

Allosteric activation of coagulation factor VIIa visualized by hydrogen exchange.

Coagulation factor VIIa (FVIIa) is a serine protease that, after binding to tissue factor (TF), plays a pivotal role in the initiation of blood coagulation. We used hydrogen exchange monitored by mass spectrometry to visualize the details of FVIIa activation by comparing the exchange kinetics of distinct molecular states, namely zymogen FVII, endoproteolytically cleaved FVIIa, TF-bound zymogen FVII, TF-bound FVIIa, and FVIIa in complex with an active site inhibitor. The hydrogen exchange kinetics of zymogen FVII and FVIIa are identical indicating highly similar solution structures. However, upon tissue factor binding, FVIIa undergoes dramatic structural stabilization as indicated by decreased exchange rates localized throughout the protease domain and in distant parts of the light chain, spanning across 50A and revealing a concerted interplay between functional sites in FVIIa. The results provide novel insights into the cofactor-induced activation of this important protease and reveal the potential for allosteric regulation in the trypsin family of proteases.

Allosteric Site↗
PubMed1978

Permease-specific mutations in Salmonella typhimurium and Escherichia coli that release the glycerol, maltose, melibiose, and lactose transport systems from regulation by the phosphoenolpyruvate:sugar phosphotransferase system.

Several carbohydrate permease systems in Salmonella typhimurium and Escherichia coli are sensitive to regulation by the phosphoenolpyruvate:sugar phosphotransferase system. Mutant Salmonella strains were isolated in which individual transport systems had been rendered insensitive to regulation by sugar substrates of the phosphotransferase system. In one such strain, glycerol uptake was insensitive to regulation; in another, the maltose transport system was resistant to inhibition; and in a third, the regulatory mutation specifically rendered the melibiose permease insensitive to regulation. An analogous mutation in E. coli abolished inhibition of the transport of beta-galactosides via the lactose permease system. The mutations were mapped near the genes which code for the affected transport proteins. The regulatory mutations rendered utilization of the particular carbohydrates resistant to inhibition and synthesis of the corresponding catabolic enzymes partially insensitive to repressive control by sugar substrates of the phosphotransferase system. Studies of repression of beta-galactosidase synthesis in E. coli were conducted with both lactose and isopropyl beta-thiogalactoside as exogenous sources of inducer. Employing high concentrations of isopropyl beta-thiogalactoside, repression of beta-galactosidase synthesis was not altered by the lactose-specific transport regulation-resistant mutation. By contrast, the more severe repression observed with lactose as the exogenous source of inducer was partially abolished by this regulatory mutation. The results support the conclusions that several transport systems, including the lactose permease system, are subject to allosteric regulation and that inhibition of inducer uptake is a primary cause of the repression of catabolic enzyme synthesis.

Biological Transport↗
PubMed2006

Expression of genes regulating malonyl-CoA in human skeletal muscle.

In humans and animal models, increased intramuscular lipid (IML) stores have been implicated in insulin resistance. Malonyl-CoA plays a critical role in cellular lipid metabolism both by serving as a precursor in the synthesis of lipids and by inhibiting lipid oxidation. In muscle, Malonyl-CoA acts primarily as a negative allosteric regulator of carnitine palmitoyl transferase-1 (CPT1) activity, thereby blocking the transport of long chain fatty acyl CoAs into the mitochondria for oxidation. In muscle, increased malonyl-CoA, decreased muscle CPT1 activity, and increased IML have all been reported in obesity. In order to determine whether malonyl-CoA synthesis might be under transcriptional as well as biochemical regulation, we measured mRNA content of several key genes that contribute to the cellular metabolism of malonyl-CoA in muscle biopsies from lean to morbidly obese subjects. Employing quantitative real-time PCR, we determined that expression of mitochondrial acetyl-CoA carboxylase 2 (ACC2) was increased by 50% with obesity (P < 0.05). In both lean and obese subjects, expression of mitochondrial ACC2 was 20-fold greater than that of cytoplasmic ACC1, consistent with their hypothesized roles in synthesizing malonyl-CoA from acetyl-CoA for CPT1 regulation and lipogenesis, respectively. In addition, in both lean and obese subjects, expression of malonyl-CoA decarboxylase was approximately 40-fold greater than fatty acid synthase, consistent with degradation, rather than lipogenesis, being the primary fate of malonyl-CoA in human muscle. No other genes showed signs of increased mRNA content with obesity, suggesting that there may be selective transcriptional regulation of malonyl-CoA metabolism in human obesity.

Acetyl-CoA Carboxylase↗
PubMed2005

Differential interaction of cardiac, skeletal muscle, and yeast tropomyosins with fluorescent (pyrene235) yeast actin.

To monitor binding of tropomyosin to yeast actin, we mutated S235 to C and labeled the actin with pyrene maleimide at both C235 and the normally reactive C374. Saturating cardiac tropomyosin (cTM) caused about a 20% increase in pyrene fluorescence of the doubly labeled F-actin but no change in WT actin C374 probe fluorescence. Skeletal muscle tropomyosin caused only a 7% fluorescence increase, suggesting differential binding modes for the two tropomyosins. The increased cTM-induced fluorescence was proportional to the extent of tropomyosin binding. Yeast tropomyosin (TPM1) produced less increase in fluorescence than did cTM, whereas that caused by yeast TPM2 was greater than either TPM1 or cTM. Cardiac troponin largely reversed the cTM-induced fluorescence increase, and subsequent addition of calcium resulted in a small fluorescence recovery. An A230Y mutation, which causes a Ca(+2)-dependent hypercontractile response of regulated thin filaments, did not change probe235 fluorescence of actin alone or with tropomyosin +/- troponin. However, addition of calcium resulted in twice the fluorescence recovery observed with WT actin. Our results demonstrate isoform-specific binding of different tropomyosins to actin and suggest allosteric regulation of the tropomyosin/actin interaction across the actin interdomain cleft.

Actins↗
PubMed1997

The AMP-activated protein kinase--fuel gauge of the mammalian cell?

A single entity, the AMP-activated protein kinase (AMPK), phosphorylates and regulates in vivo hydroxymethylglutaryl-CoA reductase and acetyl-CoA carboxylase (key regulatory enzymes of sterol synthesis and fatty acid synthesis, respectively), and probably many additional targets. The kinase is activated by high AMP and low ATP via a complex mechanism, which involves allosteric regulation, promotion of phosphorylation by an upstream protein kinase (AMPK kinase), and inhibition of dephosphorylation. This protein-kinase cascade represents a sensitive system, which is activated by cellular stresses that deplete ATP, and thus acts like a cellular fuel gauge. Our central hypothesis is that, when it detects a 'low-fuel' situation, it protects the cell by switching off ATP-consuming pathways (e.g. fatty acid synthesis and sterol synthesis) and switching on alternative pathways for ATP generation (e.g. fatty acid oxidation). Native AMP-activated protein kinase is a heterotrimer consisting of a catalytic alpha subunit, and beta and gamma subunits, which are also essential for activity. All three subunits have homologues in budding yeast, which are components of the SNF1 protein-kinase complex. SNF1 is activated by glucose starvation (which in yeast leads to ATP depletion) and genetic studies have shown that it is involved in derepression of glucose-repressed genes. This raises the intriguing possibility that AMPK may regulate gene expression in mammals. AMPK/SNF1 homologues are found in higher plants, and this protein-kinase cascade appears to be an ancient system which evolved to protect cells against the effects of nutritional or environmental stress.

AMP-Activated Protein Kinases↗
PubMed1999

Correlation between polymerizability and conformation in scallop beta-like actin and rabbit skeletal muscle alpha-actin.

In order to investigate the structural basis for functional differences among actin isoforms, we have compared the polymerization properties and conformations of scallop adductor muscle beta-like actin and rabbit skeletal muscle alpha-actin. Polymerization of scallop Ca(2+)-actin was slower than that of skeletal muscle Ca(2+)-actin. Cleavage of the actin polypeptide chain between Gly-42 and Val-43 with Escherichia coli protease ECP 32 impaired the polymerization of scallop Mg(2+)-actin to a greater extent than skeletal muscle Mg(2+)-actin. When monomeric scallop and skeletal muscle Ca(2+)-actins were subjected to limited proteolysis with trypsin, subtilisin, or ECP 32, no differences in the conformation of actin subdomain 2 were detected. At the same time, local differences in the conformations of scallop and skeletal muscle actin subdomains 1 were revealed as intrinsic fluorescence differences. Replacement of tightly bound Ca(2+) with Mg(2+) resulted in more extensive proteolysis of segment 61-69 of scallop actin than in the case of skeletal muscle actin. Furthermore, segment 61-69 was more accessible to proteolysis with subtilisin in polymerized scallop Ca(2+)-actin than in polymerized skeletal muscle Ca(2+)-actin, indicating that, in the polymeric form, the nucleotide-containing cleft is in a more open conformation in beta-like scallop actin than in skeletal muscle alpha-actin. We suggest that this difference between scallop and skeletal muscle actins is due to a less efficient shift of scallop actin subdomain 2 to the position it has in the polymer. The possible consequences of amino acid substitutions in actin subdomain 1 in the allosteric regulation of the actin cleft, and hence in the different stabilities of polymers formed by different actins, are discussed.

Actins↗
PubMed1989

Different modes of action of 3-amino-1-hydroxy-2-pyrrolidone (HA-966) and 7-chlorokynurenic acid in the modulation of N-methyl-D-aspartate-sensitive glutamate receptors.

The N-methyl-D-aspartate (NMDA)-sensitive glutamate receptors are known to be inhibited by 3-amino-1-hydroxy-2-pyrrolidone (HA-966) and 7-chlorokynurenic acid (Cl-KYN), which act at the glycine-regulated allosteric modulatory center. In this work we show that, in synaptic membranes prepared from rat brain, Cl-KYN and HA-966 inhibit the binding of [3H]glycine. Moreover, Cl-KYN can also completely inhibit the binding of [3H]glutamate to the primary transmitter recognition site for the NMDA receptor, whereas HA-966 only partially reduces this binding. Cl-KYN also abolishes the binding of the NMDA receptor antagonist [3H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP). In contrast, HA-966 increases [3H]CPP binding, affecting the affinity but not the maximal number of binding sites. This increase is inhibited by glycine and Cl-KYN. The binding of [3H] (+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine maleate (MK-801), used as an index of NMDA receptor activation, is completely inhibited by Cl-KYN but only partially by HA-966. In addition, HA-966, but not Cl-KYN, increases the potency of CPP in inhibiting [3H]MK-801 binding. Our results demonstrate that Cl-KYN and HA-966 differ in their ability to modulate the NMDA receptor, perhaps acting at distinct but overlapping recognition sites. Furthermore, our results suggest that agonist and antagonist recognition sites of the NMDA receptor may be independently regulated by glycine and HA-966, which would result, respectively, in a positive and negative allosteric modulation of the NMDA receptor complex.

Animals↗
PubMed1975

Stabilization of energy charge, generation of oscillations and multiple steady states in energy metabolism as a result of purely stoichiometric regulation.

A simple kinetic model of cell energy metabolism with autocatalytic reaction sequences has beepn analysed. The model accounts for the fact that part of energy produced in the form of ATP, or any other equivalent form, is utilized in "sparking" reactions to activate initial substrates. Analysis of the model shows that energy metabolism, in the absen-e of all non-stoichiometric (i.e. isosteric, cooperative, and allosteric) regulations, is capable of (a) stabilizing, to a high degree of accuracy, the relative concentration of the "charged form" of the energy-transferring cofactor (ATP); (b) alternating between two stable stationary states by means of hysteretic transitions; (c) generating self-oscillations in energy production. It is proposed that energy metabolism can be a source of very slow, in particular circadian (of about a one-day period), oscillations which may serve as the basis for temporal organization of the cell.

Cells↗
PubMed2001

Structural and regulatory properties of pyruvate kinase from the Cyanobacterium synechococcus PCC 6301.

Pyruvate kinase (PK) from the cyanobacterium Synechococcus PCC 6301 was purified 1,300-fold to electrophoretic homogeneity and a final specific activity of 222 micromol of pyruvate produced/min/mg of protein. The enzyme was shown to have a pI of 5.7 and to exist as a 280-kDa homotetramer composed of 66-kDa subunits. This PK appears to be immunologically related to Bacillus PK and a green algal chloroplast PK, but not to rabbit muscle PK, or vascular plant cytosolic and plastidic PKs. The N-terminal amino acid sequence of the Synechococcus PK exhibited maximal (67%) identity with the corresponding region of a putative PK-A sequence deduced from the genome of the cyanobacterium, Synechocystis PCC 6803. Synechococcus PK was relatively heat-labile and displayed a broad pH optimum around pH 7.0. Its activity was not influenced by K(+), but required high concentrations of Mg(2+), and was relatively nonspecific with respect to the nucleoside diphosphate substrate. Potent allosteric regulation by various effectors was observed (activators: hexose monophosphates, ribose 5-phosphate, glycerol 3-phosphate, and AMP; inhibitors: fructose 1,6-bisphosphate, inorganic phosphate, ATP, and several Krebs' cycle intermediates). The enzyme exhibited marked positive cooperativity for phosphoenolpyruvate, which was eliminated or reduced by the presence of the allosteric activators. The results are discussed in terms of the phylogeny and probable central role of PK in the control of cyanobacterial glycolysis.

Amino Acid Sequence↗
PubMed2005

Allosteric effects of ligands and mutations on poliovirus RNA-dependent RNA polymerase.

Protein priming of viral RNA synthesis plays an essential role in the replication of picornavirus RNA. Both poliovirus and coxsackievirus encode a small polypeptide, VPg, which serves as a primer for addition of the first nucleotide during synthesis of both positive and negative strands. This study examined the effects on the VPg uridylylation reaction of the RNA template sequence, the origin of VPg (coxsackievirus or poliovirus), the origin of 3D polymerase (coxsackievirus or poliovirus), the presence and origin of interacting protein 3CD, and the introduction of mutations at specific regions in the poliovirus 3D polymerase. Substantial effects associated with VPg origin were traced to differences in VPg-polymerase interactions. The effects of 3CD proteins and mutations at polymerase-polymerase intermolecular Interface I were most consistent with allosteric effects on the catalytic 3D polymerase molecule. In conclusion, the efficiency and specificity of VPg uridylylation by picornavirus polymerases is greatly influenced by allosteric effects of ligand binding that are likely to be relevant during the viral replicative cycle.

Allosteric Regulation↗