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PubMed2005

Structural determinants of benzodiazepine allosteric regulation of GABA(A) receptor currents.

Benzodiazepine enhancement of GABA(A) receptor current requires a gamma subunit, and replacement of the gamma subunit by the delta subunit abolishes benzodiazepine enhancement. Although it has been demonstrated that benzodiazepines bind to GABA(A) receptors at the junction between alpha and gamma subunits, the structural basis for the coupling of benzodiazepine binding to allosteric enhancement of the GABA(A) receptor current is unclear. To determine the structural basis for this coupling, the present study used a chimera strategy, using gamma2L-delta GABA(A) receptor subunit chimeras coexpressed with alpha1 and beta3 subunits in human embryonic kidney 293T cells. Different domains of the gamma2L subunit were replaced by delta subunit sequence, and diazepam sensitivity was determined. Chimeric subunits revealed two areas of interest: domain 1 in transmembrane domain 1 (M1) and domain 2 in the C-terminal portion of transmembrane domain 2 (M2) and the M2-M3 extracellular loop. In those domains, site-directed mutagenesis demonstrated that the following two groups of residues were involved in benzodiazepine transduction of current enhancement: residues Y235, F236, T237 in M1; and S280, T281, I282 in M2 as well as the entire M2-M3 loop. These results suggest that a pocket of residues may transduce benzodiazepine binding to increased gating. Benzodiazepine transduction involves a group of residues that connects the N terminus and M1, and another group of residues that may facilitate an interaction between the N terminus and the M2 and M2-M3 loop domains.

Allosteric Regulation↗
PubMed1995

Allosteric regulation of the N-methyl-D-aspartate receptor-linked ion channel complex and effects of ethanol in ethanol-withdrawal seizure-prone and -resistant mice.

The effects of ethanol, glycine, and spermidine on the specific binding of [3H]MK-801 were characterized in Triton-treated membranes prepared from the hippocampus and cortex of ethanol-withdrawal seizure-prone (WSP) and -resistant (WSR) mice. Glycine, an allosteric agonist at the NMDA receptor-linked ion channel complex, caused an increase in specific [3H]MK-801 binding to hippocampal membrane preparations. There were no significant differences in EC50 values between the selected lines for the effect of glycine (WSP, 391.7 +/- 48.4 nM; WSR, 313.4 +/- 77 nM) in the presence of 10 microM NMDA or in the maximal response to the agonist (WSP, 1.75 +/- 0.26 pmol/mg of protein; WSR, 1.67 +/- 0.22 pmol/mg of protein). The EC50 values for the spermidine-induced increase in [3H]MK-801 binding in membranes from hippocampus in the absence (WSP, 11.7 +/- 0.83 microM; WSR, 9.98 +/- 1.29 microM) or in the presence of 10 microM glycine and 10 microM NMDA (WSP, 2.1 +/- 0.35 microM; WSR, 2.37 +/- 0.42 microM) also did not differ. Similar results were obtained in cortical membranes. Saturation isotherms indicated that there was no difference in the density of [3H]MK-801 binding sites, or in their affinity for the radioligand, between the mouse lines. In addition, administration of ethanol by inhalation (24 h) to WSP and WSR mice did not cause an increase in the density of [3H]MK-801 binding sites, and there was no difference in the density or affinity of binding sites between the mouse lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed2000

UDP-N-acetylglucosamine pyrophosphorylase, a key enzyme in encysting Giardia, is allosterically regulated.

Giardia synthesizes UDP-GalNAc during cyst wall formation (encystment) via a pathway of inducible enzymes similar to that used to synthesize chitin or peptidoglycan and that includes the UTP-requiring UDP-N-acetylglucosamine pyrophosphorylase. Although it has never been reported as a regulatory enzyme in any system studied to date, kinetic data including Hill plots demonstrate clearly that UDP-N-acetylglucosamine pyrophosphorylase activity, purified from encysting Giardia, is allosterically activated anabolically by physiological levels of glucosamine 6-phosphate (3 microm). Capillary electrophoresis demonstrates that within 24 h after trophozoites are induced to encyst, the level of glucosamine 6-phosphate increases 3-fold over that of non-encysting cells and that by 48 h into encystment the level of glucosamine 6-phosphate has decreased to non-encysting levels or below. UDP-N-acetylglucosamine pyrophosphorylase protein is present constitutively in encysting as well as non-encysting cells. UDP-N-acetylglucosamine pyrophosphorylase immunoaffinity purified from encysting and non-encysting cells exhibited the same molecular weight, amino acid composition, and circular dichroism spectra. Moreover, regardless of whether the enzyme came from encysting or non-encysting cells, the change in its circular dichroism spectra and up to a 6-fold increase in its specific activity anabolically were due to its activation with glucosamine 6-phosphate. Thus, the data support the idea that UDP-N-acetylglucosamine pyrophosphorylase is a major regulatory point in amino sugar synthesis in encysting Giardia and that its allosteric anabolic activation may shift the equilibrium of this pathway toward UDP-GalNAc synthesis.

Allosteric Regulation↗
PubMed2003

Stimulation of poly(A) polymerase through a direct interaction with the nuclear poly(A) binding protein allosterically regulated by RNA.

During polyadenylation of mRNA precursors in metazoan cells, poly(A) polymerase is stimulated by the nuclear poly(A) binding protein PABPN1. We report that stimulation depends on binding of PABPN1 to the substrate RNA directly adjacent to poly(A) polymerase and results in an approximately 80-fold increase in the apparent affinity of poly(A) polymerase for RNA without significant effect on catalytic efficiency. PABPN1 associates directly with poly(A) polymerase either upon allosteric activation by oligo(A) or, in the absence of RNA, upon deletion of its N-terminal domain. The N-terminal domain of PABPN1 may function to inhibit undesirable interactions of the protein; the inhibition is relieved upon RNA binding. Tethering of poly(A) polymerase is mediated largely by the C-terminal domain of PABPN1 and is necessary but not sufficient for stimulation of the enzyme; an additional interaction dependent on a coiled-coil structure located within the N-terminal domain of PABPN1 is required for a productive interaction.

Allosteric Regulation↗
PubMed1994

Dissociation of enzyme oligomers: a mechanism for allosteric regulation.

Most enzymes exist as oligomers or polymers, and a significant subset of these (perhaps 15% of all enzymes) can reversibly dissociate and reassociate in response to an effector ligand. Such a change in subunit assembly usually is accompanied by a change in enzyme activity, providing a mechanism for regulation. Two models are described for a physical mechanism, leading to a change in activity: (1) catalytic activity depends on subunit conformation, which is modulated by subunit dissociation; and (2) catalytic or regulatory sites are located at subunit interfaces and are disrupted by subunit dissociation. Examples of such enzymes show that both catalytic sites and regulatory sites occur at the junction of 2 subunits. In addition, for 9 enzymes, kinetic studies supported the existence of a separate regulatory site with significantly different affinity for the binding of either a substrate or a product of that enzyme. Over 40 dissociating enzymes are described from 3 major metabolic areas: carbohydrate metabolism, nucleotide metabolism, and amino acid metabolism. Important variables that influence enzyme dissociation include: enzyme concentration, ligand concentration, other cellular proteins, pH, and temperature. All these variables can be readily manipulated in vitro, but normally only the first two are physiological variables. Seven of these enzymes are most active as the dissociated monomer, the others as oligomers, emphasizing the importance of a regulated equilibrium between 2 or more conformational states. Experiments to test whether enzyme dissociation occurs in vivo showed this to be the case in 6 out of 7 studies, with 4 different enzymes.

Allosteric Regulation↗
PubMedDate not supplied

Allosteric regulation of beef liver arginase activity by L-ornithine.

Inhibition of beef liver arginase by L-ornithine was investigated with two sets of independent experiments. Progress curves of the production of urea were simulated with two integrated Michaelis-Menten equations for competitive and non-competitive inhibition by ornithine. Both fitted the curves well, but failed to correctly predict the inhibition when the reaction was started with ornithine already present. Measurement of initial rates of reaction enabled an allosteric model to be built in accordance to Monod-Wyman-Changeux: arginine preferentially binds to the active state R and ornithine preferentially binds to the inactive state T. In the absence of both ligands, the R in equilibrium T equilibrium slightly favours the active state and both states bind ornithine more strongly than arginine. No great variation was observed in the 6 parameters of the model by assuming the enzyme to be a trimer or a tetramer. The model was able to predict not only the initial rate curves, from which it was derived, but also the progress curves independently obtained.

Allosteric Regulation↗
PubMed1998

Glycerol kinase from Escherichia coli and an Ala65-->Thr mutant: the crystal structures reveal conformational changes with implications for allosteric regulation.

BACKGROUND: Glycerol kinase (GK) from Escherichia coli is a velocity-modulated (V system) enzyme that has three allosteric effectors with independent mechanisms: fructose-1,6-bisphosphate (FBP); the phosphocarrier protein IIAGlc; and adenosine nucleotides. The enzyme exists in solution as functional dimers that associate reversibly to form tetramers. GK is a member of a superfamily of ATPases that share a common ATPase domain and are thought to undergo a large conformational change as an intrinsic step in their catalytic cycle. Members of this family include actin, hexokinase and the heat shock protein hsc70. RESULTS: We report here the crystal structures of GK and a mutant of GK (Ala65-->Thr) in complex with glycerol and ADP. Crystals of both enzymes contain the same 222 symmetric tetramer. The functional dimer is identical to that described previously for the IIAGlc-GK complex structure. The tetramer interface is significantly different, however, with a relative 22.3 degrees rotation and 6.34 A translation of one functional dimer. The overall monomer structure is unchanged except for two regions: the IIAGlc-binding site undergoes a structural rearrangement and residues 230-236 become ordered and bind orthophosphate at the tetramer interface. We also report the structure of a second mutant of GK (IIe474-->Asp) in complex with IIAGlc; this complex crystallized isomorphously to the wild type IIAGlc-GK complex. Site-directed mutants of GK with substitutions at the IIAGlc-binding site show significantly altered kinetic and regulatory properties, suggesting that the conformation of the binding site is linked to the regulation of activity. CONCLUSIONS: We conclude that the new tetramer structure presented here is an inactive form of the physiologically relevant tetramer. The structure and location of the orthophosphate-binding site is consistent with it being part of the FBP-binding site. Mutational analysis and the structure of the IIAGlc-GK(IIe474-->Asp) complex suggest the conformational transition of the IIAGlc-binding site to be an essential aspect of IIAGlc regulation.

Alanine↗
PubMed1999

Characterization of ADP-glucose pyrophosphorylase from Rhodobacter sphaeroides 2.4.1: evidence for the involvement of arginine in allosteric regulation.

ADP-glucose pyrophosphorylase (ADPGlc PPase, EC 2.7.7.27) from Rhodobacter sphaeroides 2.4.1 has been purified to near homogeneity. The enzyme reacted in Western blots to polyclonal antibodies raised against other bacterial ADPGlc PPases. The purified enzyme was found to be activated by fructose 6-phosphate, fructose 1,6-bisphosphate, and pyruvate and inhibited by phosphate, phosphoenolpyruvate, ADP, and pyridoxal phosphate. Kinetic studies indicate that AMP, while having little effect on kinetic parameters at pH 8 in the absence of effectors, is a specific ligand for an allosteric site(s). Treatment of the purified enzyme with the arginyl reagents 2,3-butanedione and phenylglyoxal resulted in desensitization of the enzyme to both activation and inhibition by metabolites. Phosphate, fructose 6-phosphate, and AMP were found to protect the enzyme against allosteric desensitization supportive of these metabolites interacting at common site(s) or with a common enzyme form. As a first step in cloning the gene coding for this enzyme, a polymerase chain reaction fragment was generated from genomic DNA using primers based on amino terminal sequencing data and a highly conserved region in known ADPGlc PPases. The sequence of this fragment and position of amino terminal arginines in comparison to other known ADPGlc PPases is discussed in relation to the kinetic and chemical modification data.

Allosteric Regulation↗
PubMed1990

Evidence for the allosteric regulation of the mitochondrial K+/H+ antiporter by matrix protons.

It is well accepted that the mitochondrial K+/H+ antiporter is regulated by matrix Mg2+; however, this is not the only factor controlling its activity. The precise conditions used to deplete divalent cations have profound effects on the subsequent activity of the antiporter in a KOAc assay medium. Examination of the proton fluxes during both pretreatment and subsequent assay of K+/H+ antiport reveals that differences in K+/H+ antiport activity correlate very well with differences in matrix pH. Thus, inhibition of the K+/H+ antiporter following depletion of Mg2+ appears to result from inhibition by matrix protons. To test this hypothesis, we have examined the effect of modulating matrix pH in three different ways on the activity of the K+/H+ antiporter: 1) lowering the pH of the K+ pretreatment medium to 6.7 leads to inactivation of the K+/H+ antiporter; 2) adding NH4+ to the assay medium eliminates the lag in activity induced by depleting Mg2+ in a pretreatment medium containing NH4+; 3) permitting mitochondria to respire in a tetraethylammonium(+)-containing pretreatment medium activates the K+/H+ antiporter. Each one of these procedures leads to a change in matrix pH and an effect on K+/H+ antiport which appears to require regulation of the K+/H+ antiporter by matrix protons. This finding is not only physiologically significant but also provides a useful definition of conditions required for unmasking the K+/H+ antiporter in a reproducible manner.

Acetates↗
PubMed2005

Role of Cys-295 on subunit interactions and allosteric regulation of phosphofructokinase-2 from Escherichia coli.

In a previous work, chemical modification of Cys-238 of Escherichia coli Pfk-2 raised concerns on the importance of the dimeric state of Pfk-2 for enzyme activity, whereas modification of Cys-295 impaired the enzymatic activity and the MgATP-induced tetramerization of the enzyme. The results presented here demonstrate that the dimeric state of Pfk-2 is critical for the stability and the activity of the enzyme. The replacement of Cys-238 by either Ala or Phe shows no effect on the kinetic parameters, allosteric inhibition, dimer stability and oligomeric structure of Pfk-2. However, the mutation of Cys-295 by either Ala or Phe provokes a decrease in the k(cat) value and an increment in the K(m) values for both substrates. We suggest that the Cys-295 residue participates in intersubunit interactions in the tetramer since the Cys-295-Phe mutant exhibits higher tetramer stability, which in turn results in an increase in the fructose-6-P concentration required for the reversal of the MgATP inhibition relative to the wild type enzyme.

Allosteric Regulation↗
PubMed2004

Crystal structure of the reaction complex of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Thermotoga maritima refines the catalytic mechanism and indicates a new mechanism of allosteric regulation.

3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) catalyzes the first reaction of the aromatic biosynthetic pathway in bacteria, fungi, and plants, the condensation of phosphoenolpyruvate (PEP) and d-erythrose-4-phosphate (E4P) with the formation of DAHP. Crystals of DAHPS from Thermotoga maritima (DAHPS(Tm)) were grown in the presence of PEP and metal cofactor, Cd(2+), and then soaked with E4P at 4 degrees C where the catalytic activity of the enzyme is negligible. The crystal structure of the "frozen" reaction complex was determined at 2.2A resolution. The subunit of the DAHPS(Tm) homotetramer consists of an N-terminal ferredoxin-like (FL) domain and a (beta/alpha)(8)-barrel domain. The active site located at the C-end of the barrel contains Cd(2+), PEP, and E4P, the latter bound in a non-productive conformation. The productive conformation of E4P is suggested and a catalytic mechanism of DAHPS is proposed. The active site of DAHPS(Tm) is nearly identical to the active sites of the other two known DAHPS structures from Escherichia coli (DAHPS(Ec)) and Saccharomyces cerevisiae (DAHPS(Sc)). However, the secondary, tertiary, and quaternary structures of DAHPS(Tm) are more similar to the functionally related enzyme, 3-deoxy-d-manno-octulosonate-8-phosphate synthase (KDOPS) from E.coli and Aquiflex aeolicus, than to DAHPS(Ec) and DAHPS(Sc). Although DAHPS(Tm) is feedback-regulated by tyrosine and phenylalanine, it lacks the extra barrel segments that are required for feedback inhibition in DAHPS(Ec) and DAHPS(Sc). A sequence similarity search revealed that DAHPSs of phylogenetic family Ibeta possess a FL domain like DAHPS(Tm) while those of family Ialpha have extra barrel segments similar to those of DAHPS(Ec) and DAHPS(Sc). This indicates that the mechanism of feedback regulation in DAHPS(Tm) and other family Ibeta enzymes is different from that of family Ialpha enzymes, most likely being mediated by the FL domain.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed2005

Allosteric regulation of the higher plant ADP-glucose pyrophosphorylase is a product of synergy between the two subunits.

The higher plant ADP-glucose pyrophosphorylase (AGPase) is a heterotetramer consisting of two regulatory large subunits (LSs) and two catalytic small subunits (SSs). To further characterize the roles of these subunits in determining enzyme function, different combinations of wildtype LS (LWT) and variant forms (LUpReg1, LM345) were co-expressed with wildtype SS (SWT) and variant forms (STG-15 and Sdevo330) and their enzyme properties compared to those measured for the heterotetrameric wildtype enzyme and SS homotetrameric enzymes. Analysis of the allosteric regulatory properties of the various enzymes indicates that although the LS is required for optimal activation by 3-phosphoglyceric acid and resistance to Pi, the overall allosteric regulatory and kinetic properties are specified by both subunits. Our results show that the regulatory and kinetic properties of AGPase are not simply due to the LS modulating the properties of the SS but, instead, are a product of synergistic interaction between the two subunits.

Allosteric Regulation↗
PubMed1989

Allosteric regulation of the state of adenylylation of glutamine synthetase in permeabilized cell preparations of Rhodopseudomonas sphaeroides.

Following a freeze-thaw cycle, and the treatment of Rhodopseudomonas sphaeroides with the nonionic detergent Lubrol PX, the permeabilized cell suspensions can be assayed directly both for the intracellular levels of glutamine synthetase and the state of adenylylation (i.e. the average number n of adenylylated subunits/dodecameric molecules). It seems that all components of the bicycle system are retained if cells grown with low concentrations of ammonia as the sole nitrogen source are used. The value of n was dependent upon the concentration of substrates (ATP, Pi) and allosteric effectors (ATP, glutamine and alpha-ketoglutarate) of adenylytransferase. The value of n affected by UTP, the specific substrate of the uridylyltransferase shows first the evidence that the bicycle cascade control system studied in Escherichia coli may exist in this phototrophic bacterium.

Allosteric Regulation↗
PubMed2006

Allosteric regulation of Hsp70 chaperones involves a conserved interdomain linker.

The 70-kDa heat shock proteins (Hsp70) are essential members of the cellular chaperone machinery that assists protein-folding processes. To perform their functions Hsp70 chaperones toggle between two nucleotide-controlled conformational states. ATP binding to the ATPase domain triggers the transition to the low affinity state of the substrate-binding domain, while substrate binding to the substrate-binding domain in synergism with the action of a J-domain-containing cochaperone stimulates ATP hydrolysis and thereby transition to the high affinity state. Thus, ATPase and substrate-binding domains mutually affect each other through an allosteric control mechanism, the basis of which is largely unknown. In this study we identified two positively charged, surface-exposed residues in the ATPase domain and a negatively charged residue in the linker connecting both domains that are important for interdomain communication. Furthermore, we demonstrate that the linker alone is sufficient to stimulate the ATPase activity, an ability that is lost upon amino acid replacement. The linker therefore is most likely the lever that is wielded by the substrate-binding domain and the cochaperone onto the ATPase domain to induce a conformation favorable for ATP hydrolysis. Based on our results we propose a mechanism of interdomain communication.

Adenosine Triphosphatases↗
PubMed1997

Allosteric regulation of RecA protein function is mediated by Gln194.

Binding of ATP to the RecA protein induces a high affinity DNA binding required for activation of enzyme function. Screens for in vivo recombination and repressor cleavage activities show Gln194 to be intolerant of all substitutions. Analyses of three mutant proteins (Q194N, Q194E, and Q194A) show that although basal enzyme function is maintained, each protein no longer displays an ATP-induced increase in DNA binding affinity. High salt activation of RecA function is also disrupted by these mutations. In contrast, ATP-induced changes in the oligomeric structure of RecA are maintained in the mutant proteins. These results demonstrate that Gln194 is a critical "allosteric switch" for ATP-induced activation of RecA function but is not the exclusive mediator of ATP-induced changes in RecA.

Adenosine Triphosphatases↗
PubMed1996

Allosteric regulation of liver phosphorylase a: revisited under approximated physiological conditions.

Phosphorylase removes glucosyl units from the terminal branches of glycogen through phosphorolysis, forming glucose-1-P. It is present in two interconvertible forms, phosphorylase a and b. The a form is the active form and is rate limiting in glycogen degradation. The activities of phosphorylase a and of total phosphorylase as conventionally measured exceed the activities of glycogen synthase R (active form) and of total synthase by approximately 10- and 20-fold. Thus, unless phosphorylase a is inhibited or compartmentalized or its substrates are exceedingly low in vivo, net glycogen synthesis could not occur. In addition, following an administered dose of glucose, phosphorylase a activity changes little when glycogen is being synthesized, is stable, or is being degraded, suggesting an important role for allosteric effectors in regulation. Therefore, we have determined the effect of potential modifiers of enzyme activity at estimated intracellular concentrations. Purified liver phosphorylase a was used. Activity was measured in the direction of glycogenolysis, at 37 degrees C, pH 7.0, and under initial rate conditions. Both a Km and a near-saturating concentration of inorganic phosphate (substrate) were used in the assays. A physiological concentration of AMP was saturating. It decreased the Km for Pi by approximately 50% and stimulated activity. ADP, ATP, and glucose inhibited activity. Fructose-1-P inhibited activity only at a high and nonphysiological concentration. Glucose-6-P and UDP-glucose were not significant inhibitors. Inhibition of activity by ADP was little affected by the addition of AMP. However, AMP partially abolished the inhibitory effect of ATP and completely abolished the inhibitory effect of glucose. When AMP, ADP, ATP, glucose-6-P, UDP-glucose, glucose, and fructose-1-P were added together, the net effect was no change in phosphorylase a activity compared to the activity without any effectors. In addition, changes in glucose concentration did not affect activity. K glutamine modestly stimulated activity. Numerous other metabolites were tested and were without effect. The present data indicate that the known endogenous allosteric effectors cannot explain the smaller than expected in vivo phosphorylase a activity or the regulation of phosphorylase a activity.

Adenine Nucleotides↗
PubMed1998

Allosteric regulation and substrate channeling in multifunctional pyrimidine biosynthetic complexes: analysis of isolated domains and yeast-mammalian chimeric proteins.

The initial steps of pyrimidine biosynthesis in yeast and mammals are catalyzed by large multifunctional proteins of similar size, sequence and domain structure, but appreciable functional differences. The mammalian protein, CAD, has carbamyl phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) activities. The yeast protein, ura2, catalyzes the first two reactions and has a domain, called pDHO, which is homologous to mammalian DHOase, but is inactive. In CAD, only CPSase is regulated, whereas both CPSase and ATCase in the yeast protein are inhibited by UTP. These functional differences were explored by constructing a series of mammalian yeast chimeras. The isolated ATCase domain is catalytically active, but is not regulated. The inclusion of the yeast sequences homologous to the mammalian regulatory domain (B3) and the intervening pDHO domain did not confer regulation. Chimeric proteins in which the homologous regions of the mammalian protein were replaced by the corresponding domains of ura2 exhibited full catalytic activity, as well regulation of the CPSase, but not the ATCase, activities. The yeast B3 subdomain confers UTP sensitivity on the mammalian CPSase, suggesting that it is the locus of CPSase regulation in ura2. Taken together, these results indicate that there are regulatory site(s) in ura2. Channeling is impaired in all the chimeric complexes and completely abolished in the chimera in which the pDHO domain of yeast is replaced by the mammalian DHO domain.

Allosteric Regulation↗