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PubMed1999

Cannabinoid agonists and antagonists discriminated by receptor binding in rat cerebellum.

1. The effect of allosteric regulators on the binding affinity of a number of cannabinoid receptor ligands of varying efficacy in the rat cerebellum was investigated. 2. Radioligand ([3H]-SR141716A) competition curves were constructed in the presence or absence of sodium ions, magnesium ions and guanine nucleotides. 3. It was found that the presence of these allosteric regulators did not affect the affinity of the two antagonists used but did cause a significant decrease in the affinity of full and partial agonists. 4. This reduction in affinity ranged from a 3.67 fold rightward shift of the displacement curve of a mixed agonist/antagonist (3-(6-cyano-2-hexynyl)-delta-8-tetrahydrocannabinol-O-823) to a 38 fold rightward shift for 3-(1, 1-dimethyl-6-dimethylcarboxamide)-delta-8-tetrahydrocannabinol (O-1125), a full agonist. 5. In summary, the results of this study suggest a simple method for the inference of functional data using the classical radioligand binding assay.

Allosteric Regulation↗
PubMedDate not supplied

The effect of inositol hexaphosphate in the high-affinity hemoglobin of the Andean chicken (Gallus gallus).

A strain of high-altitude chickens (Gallus gallus) with a high hemoglobin-oxygen affinity has been found in the Peruvian Andes. The purpose in this work was to investigate the relation of this high affinity with the allosteric regulation of the inositol hexaphosphate effector (IHP). The hemoglobin-oxygen affinity has been determined in phosphate-free hemoglobin solutions before and after the addition of IHP. The results showed that the addition of IHP to the phosphate-free hemoglobin solutions increased the affinity significantly less in the high-altitude chicken (26.3 Torr) than in the sea-level chicken (42.2 Torr) (P < 0.001). It was concluded that the high-affinity hemoglobin is probably dependent on a molecular change affecting the allosteric regulation, although a change in the intrinsic properties of the hemoglobin cannot be discarded.

Adaptation, Physiological↗
PubMed1996

Site-directed mutagenesis of the human A1 adenosine receptor: influences of acidic and hydroxy residues in the first four transmembrane domains on ligand binding.

To provide new insights into ligand/A1 adenosine receptor (A1 AR) interactions, site-directed mutagenesis was used to test the role of several residues in the first four transmembrane (TM) domains of the human A1 AR. Based on multiple sequence analysis of all known ARs, both acidic (glutamic acid and aspartic acid) and polar hydroxy (serine and threonine) amino acids were identified that could potentially play a role in binding adenosine. Glu16 (TM1), Asp55 (TM2), Ser93 and Ser94 (TM3), Ser135 (TM4), and Thr 141 (TM4) were identified in all ARs, and Ser6 and Ser23 (TM1) were identified in all A1 ARs. To test the role of these residues, each was individually mutated to alanine. When Ala6, Ala23, Ala50, Ala93, Ala135, and Ala141 constructs were tested, affinities for [3H]2-chloro-N6-cyclopentyladenosine (CCPA) and [3H]1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were similar to those seen for the wild-type receptor. After conversion of Glu16 to Ala16, the affinity for [3H]CCPA and other agonists fell 10-100-fold, whereas the affinity for [3H]DPCPX and other antagonists was not affected. After conversion of Asp55 to Ala55, the affinity for [3H]CCPA and other agonists increased < or = 100-fold, whereas the affinity for [3H]DPCPX and other antagonists was not affected. Studies of the Ala55 construct also revealed that Asp55 is responsible for allosteric regulation of binding by sodium because the affinity for [3H]CCPA did not change over broad ranges of sodium concentrations. When Ser94 was converted to Ala94, A1 AR immunoreactivity was present on stable cell lines; however, functional binding sites could not be detected. When Ser94 was converted to Thr94, the affinity for some xanthine antagonists fell. These data show that Glu16 in TM1 and Asp55 in TM2 play important roles in agonist/A1 AR interactions and show that Asp55 is responsible for allosteric regulation of ligand/A1 AR binding by sodium. We also identify Ser94 as an important site for ligand binding.

Allosteric Regulation↗
PubMed1999

Quantum mechanical analysis of oxygenated and deoxygenated states of hemocyanin: theoretical clues for a plausible allosteric model of oxygen binding.

In this work with ab initio computations, we describe relevant interactions between protein active sites and ligands, using as a test case arthropod hemocyanins. A computational analysis of models corresponding to the oxygenated and deoxygenated forms of the hemocyanin active site is performed using the Density Functional Theory approach. We characterize the electron density distribution of the binding site with and without bound oxygen in relation to the geometry, which stems out of the crystals of three hemocyanin proteins, namely the oxygenated form from the horseshoe crab Limulus polyphemus, and the deoxygenated forms, respectively, from the same source and from another arthropod, the spiny lobster Panulirus interruplus. Comparison of the three available crystals indicate structural differences at the oxygen binding site, which cannot be explained only by the presence and absence of the oxygen ligand, since the geometry of the ligand site of the deoxygenated Panulirus hemocyanin is rather similar to that of the oxygenated Limulus protein. This finding was interpreted in the frame of a mechanism of allosteric regulation for oxygen binding. However, the cooperative mechanism, which is experimentally well documented, is only partially supported by crystallographic data, since no oxygenated crystal of Panulirus hemocyanin is presently available. We address the following question: is the local ligand geometry responsible for the difference of the dicopper distance observed in the two deoxygenated forms of hemocyanin or is it necessary to advocate the allosteric regulation of the active site conformations in order to reconcile the different crystal forms? We find that the difference of the dicopper distance between the two deoxygenated hemocyanins is not due to the small differences of ligand geometry found in the crystals and conclude that it must be therefore stabilized by the whole protein tertiary structure.

Allosteric Regulation↗
PubMed1999

Glucose catabolism of Escherichia coli strains with increased activity and altered regulation of key glycolytic enzymes.

This study investigates the effect of overexpression of key glycolytic enzymes exhibiting either native or alternative allosteric regulation on glucose bioconversion by resting Escherichia coli cells previously engineered for ethanol production. Homologous and heterologous pyruvate kinases (Pyk) and phosphofructokinases (Pfk) were individually and simultaneously overexpressed. Overexpression of the E. coli Pfk led to a shift from ethanol to lactate formation (three-fold above the control level) while overexpression of Pyks accelerated lactate formation two-fold with less reduction in ethanol formation. Further increase in lactate formation (five-fold above the control level) resulted from overexpression of Pfk from Lactobacillus bulgaricus which, unlike the E. coli Pfk, is not allosterically regulated by either phosphoenolpyruvate or ADP. These effects on the carbon flux distribution were accompanied by significant changes in the intracellular concentrations of several glycolytic intermediates. Increased Pfk levels led primarily to reduced levels of hexose phosphates. Increased Pyk activity resulted in more complex changes which were different for overexpressed native Pyk and for overexpressed Bacillus stearothermophilus Pyk, which differs from E. coli Pyk in lacking activation by fructose 1,6-diphosphate, but is allosterically activated by AMP and ribose 5-phosphate. Simultaneous overexpression of native Pfk and Pyk caused a Pfk-overexpression-like phenotype with lower levels of hexose phosphates and further increased lactate formation (nine-fold above the control level). The flux data demonstrate that overexpression of even single enzymes early in a central pathway can increase the fluxes to a particular metabolic product, although it may not affect the glucose uptake rate.

Cloning, Molecular↗
PubMed2001

Structural and functional linkages between subunit interfaces in mammalian pyruvate kinase.

Mammalian pyruvate kinase (PK) is a four-domain enzyme that is active as a homo-tetramer. Tissue-specific isozymes of PK exhibit distinct levels of allosteric regulation. PK expressed in muscle tissue (M1-PK) shows hyperbolic steady-state kinetics, whereas PK expressed in kidney tissue (M2-PK) displays sigmoidal kinetics. Rabbit M1 and M2-PK are isozymes whose sequences differ in only 22 out of 530 residues per subunit, and these changes are localized in an inter-subunit interface. Previous studies have shown that a single amino acid mutation to M1-PK at either the Y (S402P) or Z (T340 M) subunit interface can confer a level of allosteric regulation that is intermediate to M1-PK and M2-PK. In an effort to elucidate the roles of the inter-subunit interaction in signal transmission and the functional/structural connectivity between these interfaces, the S402P mutant of M1-PK was crystallized and its structure resolved to 2.8 A. Although the overall S402P M1-PK structure is nearly identical with the wild-type structure within experimental error, significant differences in the conformation of the backbone are found at the site of mutation along the Y interface. In addition, there is a significant change along the Z interface, namely, a loss of an inter-subunit salt-bridge between Asp177 of domain B and Arg341 of domain A of the opposing subunit. Concurrent with the loss of the salt-bridge is an increase in the degree of rotational flexibility of domain B that constitutes the active site. Comparison of previous PK structures shows a correlation between an increase in this domain movement with the loss of the Asp177: Arg341 salt-bridge. These results identify the structural linkages between the Y and Z interfaces in regulating the interconversion of conformational states of rabbit M1-PK.

Allosteric Regulation↗
PubMed1999

ATP regulation of type 1 inositol 1,4,5-trisphosphate receptor channel gating by allosteric tuning of Ca(2+) activation.

Inositol 1,4,5-trisphosphate (InsP(3)) mobilizes intracellular Ca(2+) by binding to its receptor (InsP(3)R), an endoplasmic reticulum-localized Ca(2+) release channel. Patch clamp electrophysiology of Xenopus oocyte nuclei was used to study the effects of cytoplasmic ATP concentration on the cytoplasmic Ca(2+) ([Ca(2+)](i)) dependence of single type 1 InsP(3)R channels in native endoplasmic reticulum membrane. Cytoplasmic ATP free-acid ([ATP](i)), but not the MgATP complex, activated gating of the InsP(3)-liganded InsP(3)R, by stabilizing open channel state(s) and destabilizing the closed state(s). Activation was associated with a reduction of the half-maximal activating [Ca(2+)](i) from 500 +/- 50 nM in 0 [ATP](i) to 29 +/- 4 nM in 9.5 mM [ATP](i), with apparent ATP affinity = 0.27 +/- 0.04 mM, similar to in vivo concentrations. In contrast, ATP was without effect on maximum open probability or the Hill coefficient for Ca(2+) activation. Thus, ATP enhances gating of the InsP(3)R by allosteric regulation of the Ca(2+) sensitivity of the Ca(2+) activation sites of the channel. By regulating the Ca(2+)-induced Ca(2+) release properties of the InsP(3)R, ATP may play an important role in shaping cytoplasmic Ca(2+) signals, possibly linking cell metabolic state to important Ca(2+)-dependent processes.

Adenosine Triphosphate↗
PubMed2006

Crystallographic identification of an ordered C-terminal domain and a second nucleotide-binding site in RecA: new insights into allostery.

RecA protein is a crucial and central component of the homologous recombination and DNA repair machinery. Despite numerous studies on the protein, several issues concerning its action, including the allosteric regulation mechanism have remained unclear. Here we report, for the first time, a crystal structure of a complex of Mycobacterium smegmatis RecA (MsRecA) with dATP, which exhibits a fully ordered C-terminal domain, with a second dATP molecule bound to it. ATP binding is an essential step for all activities of RecA, since it triggers the formation of active nucleoprotein filaments. In the crystal filament, dATP at the first site communicates with a dATP of the second site of an adjacent subunit, through conserved residues, suggesting a new route for allosteric regulation. In addition, subtle but definite changes observed in the orientation of the nucleotide at the first site and in the positions of the segment preceding loop L2 as well as in the segment 102-105 situated between the 2 nt, all appear to be concerted and suggestive of a biological role for the second bound nucleotide.

Allosteric Regulation↗
PubMed1995

Allosteric linkages between beta-site covalent transformations and alpha-site activation and deactivation in the tryptophan synthase bienzyme complex.

This work examines two aspects of the catalytic mechanism and allosteric regulation of the tryptophan synthase bienzyme complex from Salmonella typhimurium: (a) the chemical mechanism by which indole and other nucleophiles react with the enzyme-bound alpha-aminoacrylate Schiff base intermediate, E(A-A), to form quinonoidal intermediates, E(Q), and (b) the effects of covalent transformations at the beta-site on the catalytic activity of the alpha-site. Transient kinetic studies in combination with alpha-secondary deuterium isotope effects are undertaken to determine the mechanism of nucleophile addition to E(A-A). These studies establish that nucleophilic attack is best described by a two-step reaction sequence consisting of a binding step that is followed by Michael addition to the conjugated double bond of E(A-A). Analysis of isotope effects suggests that the transition state for indole addition gives an E(A-A) beta-carbon that resembles an sp3 center, while the stronger nucleophiles, indoline and beta-mercaptoethanol, have transition states that appear to more closely resemble an sp2 beta-carbon. The effects of beta-site covalent transformations on alpha-site catalysis were studied using quasi-stable beta-site intermediates and the alpha-site substrate analogue 3-[6-nitroindole]-D-glycerol 3'-phosphate (6-nitro-IGP). It was found that the cleavage of 6-nitro-IGP is strongly activated by the formation of E(A-A) and various E(Q) species at the beta-site but not by external aldimine species. Therefore, we conclude that the conversion of the L-Ser external aldimine to E(A-A) is the beta-site process which activates the alpha-site, while conversion of E(Q) to the L-Trp external aldimine triggers deactivation of the alpha-site. These findings are discussed within the context of allosteric regulation of substrate channeling in tryptophan synthase catalysis.

Allosteric Regulation↗
PubMed2000

Kinetic and structural analysis of the ultrasensitive behaviour of cyanobacterial ADP-glucose pyrophosphorylase.

The kinetic and (supra)molecular properties of the ultrasensitive behaviour of ADP-glucose pyrophosphorylase (AGPase) from Anabaena PCC 7120 (a cyanobacterium) were exhaustively studied. The response of the enzyme toward the allosteric activator 3-phosphoglycerate (3PGA) occurs with ultrasensitivity as a consequence of the cross-talk with the inhibitor P(i). Molecular 'crowding' renders AGPase more sensitive to the interplay between the allosteric regulators and, consequently, enhances the ultrasensitive response. In crowded media, and when orthophosphate is present, the activation kinetics of the enzyme with 3PGA proceed with increased co-operativity and reduced affinity toward the activator. Under conditions of ultrasensitivity, the enzyme's maximal activation takes place in a narrow range of 3PGA concentrations. Moreover, saturation kinetics of the enzyme with respect to its substrates, glucose 1-phosphate and ATP, were different at low or high 3PGA levels in crowded media. Only under the latter conditions did AGPase exhibit discrimination between low or high levels of the activator, which increased the affinity toward the substrates and the maximal activity reached by the enzyme. Studies of fluorescence emission of tryptophan residues, fourth-derivative spectroscopy and size-exclusion chromatography indicated that the ultrasensitive behaviour is correlated with intramolecular conformational changes induced in the tertiary structure of the homotetrameric enzyme. The results suggest a physiological relevance of the ultrasensitive response of AGPase in vivo, since the enzyme could be subtly sensing changes in the levels of allosteric regulators and substrates, and thus determining the flux of metabolites toward synthesis of storage polysaccharides.

Allosteric Regulation↗
PubMed2005

Identification of the geometric requirements for allosteric communication between the alpha- and beta-subunits of tryptophan synthase.

The pyridoxal 5'-phosphate-dependent tryptophan synthase alpha2beta2 complex is a paradigmatic protein for substrate channeling and allosteric regulation. The enzymatic activity is modulated by a ligand-mediated equilibrium between open (inactive) and closed (active) conformations of the alpha- and beta-subunit, predominantly involving the mobile alpha loop 6 and the beta-COMM domain that contains beta helix 6. The alpha ligand-triggered intersubunit communication seems to rely on a single hydrogen bond formed between the carbonyl oxygen of betaSer-178 of beta helix 6 and the NH group of alphaGly-181 of alpha loop 6. We investigated whether and to what extent mutations of alphaGly-181 and betaSer-178 affect allosteric regulation by the replacement of betaSer-178 with Pro or Ala and of alphaGly-181 with either Pro to remove the amidic proton that forms the hydrogen bond or Ala, Val, and Phe to analyze the dependence on steric hindrance of the open-closed conformational transition. The alpha and beta activity assays and the equilibrium distribution of beta-subunit catalytic intermediates indicate that mutations do not significantly influence the intersubunit catalytic activation but completely abolish ligand-induced alpha-to beta-subunit signaling, demonstrating distinct pathways for alpha-beta-site communication. Limited proteolysis experiments indicate that the removal of the interaction between betaSer-178 and alphaGly-181 strongly favors the more trypsin-accessible open conformation of the alpha-active site. When the hydrogen bond cannot be formed, the alpha-subunit is unable to attain the closed conformation, and consequently, the allosteric signal is aborted at the subunit interface.

Allosteric Regulation↗
PubMed1995

The dihedral symmetry of the p53 tetramerization domain mandates a conformational switch upon DNA binding.

The p53 tumor suppressor forms stable tetramers, whose DNA binding activity is allosterically regulated. The tetramerization domain is contained within the C-terminus (residues 323-355) and its three-dimensional structure exhibits dihedral symmetry, such that a p53 tetramer can be considered a dimer of dimers. Under conditions where monomeric p53 fails to bind DNA, we studied the effects of p53 C-terminal mutations on DNA binding. Residues 322-355 were sufficient to drive DNA binding of p53 as a tetramer. Within this region residues predicted by the three-dimensional structure to stabilize tetramerization, such as Arg337 and Phe341, were critical for DNA binding. Furthermore, substitution of Leu344 caused p53 to dissociate into DNA binding-competent dimers, consistent with the location of this residue at the dimer-dimer interface. The p53 DNA site contains two inverted repeats juxtaposed to a second pair of inverted repeats. Thus, the four repeats exhibit cyclic-translation symmetry and cannot be recognized simultaneously by four dihedrally symmetric p53 DNA binding domains. The discrepancy may be resolved by flexible linkers between the p53 DNA binding and tetramerization domains. When these linkers were deleted p53 exhibited novel DNA binding properties consistent with an inability to recognize four contiguous DNA repeats. Allosteric regulation of p53 DNA binding may involve repositioning the DNA binding domains from a dihedrally symmetric state to a DNA-bound asymmetric state.

Allosteric Regulation↗
PubMed1999

Regulation of the catalytic function of coagulation factor VIIa by a conformational linkage of surface residue Glu 154 to the active site.

Coagulation factor VIIa is an allosterically regulated trypsin-like serine protease that initiates the coagulation pathways upon complex formation with its cellular receptor and cofactor tissue factor (TF). The analysis of a conformation-sensitive monoclonal antibody directed to the macromolecular substrate exosite in the VIIa protease domain demonstrated a conformational link from this exosite to the catalytic cleft that is independent of cofactor-induced allosteric changes. In this study, we identify Glu 154 as a critical surface-exposed exosite residue side chain that undergoes conformational changes upon active site inhibitor binding. The Glu 154 side chain is important for hydrolysis of scissile bond mimicking peptidyl p-nitroanilide substrates, and for inhibition of VIIa's amidolytic function upon antibody binding. This exosite residue is not linked to the catalytic cleft residue Lys 192 which plays an important role in thrombin's allosteric coupling to exosite I. Allosteric linkages between VIIa's active site and the cofactor binding site or between the cofactor binding site and the macromolecular substrate exosite were not influenced by mutation of Glu 154. Glu 154 thus only influences the linkage of the macromolecular substrate binding exosite to the catalytic center. These data provide novel evidence that allosteric regulation of VIIa's catalytic function involves discrete and independent conformational linkages and that allosteric transitions in the VIIa protease domain are not globally coupled.

Antibodies, Monoclonal↗
PubMed2002

Alleviation of intrasteric inhibition by the pathogenic activation domain mutation, D444N, in human cystathionine beta-synthase.

Human cystathionine beta-synthase is a heme protein that catalyzes the condensation of serine and homocysteine to form cystathionine in a pyridoxal phosphate-dependent reaction. Mutations in this enzyme are the leading cause of hereditary hyperhomocysteinemia with attendant cardiovascular and other complications. The enzyme is activated approximately 2-fold by the allosteric regulator S-adenosylmethionine (AdoMet), which is presumed to bind to the C-terminal regulatory domain. The regulatory domain exerts an inhibitory effect on the enzyme, and its deletion is correlated with a 2-fold increase in catalytic activity and loss of responsiveness to AdoMet. A mutation in the C-terminal regulatory domain, D444N, displays high levels of enzyme activity, yet is pathogenic. In this study, we have characterized the biochemical penalties associated with this mutation and demonstrate that it is associated with a 4-fold lower steady-state level of cystathionine beta-synthase in a fibroblast cell line that is homozygous for the D444N mutation. The activity of the recombinant D444N enzyme mimics the activity of the wild-type enzyme seen in the presence of AdoMet and can be further activated approximately 2-fold in the presence of supraphysiolgical concentrations of the allosteric regulator. The mutation increases the K(act) for AdoMet from 7.4 +/- 0.2 to 460 +/- 130 microM, thus rendering the enzyme functionally unresponsive to AdoMet under physiological concentrations. These results indicate that the D444N mutation partially abrogates the intrasteric inhibition imposed by the C-terminal domain. We propose a model that takes into account the three kinetically distinguishable states that are observed with human cystathionine beta-synthase: "basal" (i.e., wild-type enzyme as isolated), "activated" (wild-type enzyme + AdoMet or the D444N mutant as isolated), and superactivated (D444N mutant + AdoMet or wild-type enzyme lacking the C-terminal regulatory domain).

Allosteric Regulation↗
PubMed2002

Kinetic and allosteric consequences of mutations in the subunit and domain interfaces and the allosteric site of yeast pyruvate kinase.

The mechanism by which pyruvate kinase (PK) is allosterically activated by fructose-1,6-bisphosphate (FBP) is poorly understood. To identify residues key to allostery of yeast PK, a point mutation strategy was used. T403E and R459Q mutations in the FBP binding site caused reduced FBP affinity. Introducing positive charges at the 403, 458, and 406 positions in the FBP binding site had little consequence. The mutation Q299N in the A [bond] A subunit interface caused the enzyme response to ADP to be sensitive to FBP. The T311M A [bond] A interface mutant has a decreased affinity for PEP and FBP, and is dependent on FBP for activity. The R369A mutation in the C [bond] C interface only moderately influenced allostery. Creating an E392A mutation in the C [bond] C subunit interface eliminated all cooperativity and allosteric regulation. None of the seven A [bond] C domain interface mutations altered allostery. A model that includes a central role for E392 in allosteric regulation of yeast PK is proposed.

Adenosine Diphosphate↗
PubMed1998

Skeletal muscle glycolytic capacity and phosphofructokinase regulation in horses with polysaccharide storage myopathy.

OBJECTIVE: To determine whether polysaccharide storage myopathy (PSSM) in Quarter Horses is attributable to a defect in glycolysis or in the allosteric regulation of phosphofructokinase (PFK) enzyme. ANIMALS: Muscle biopsy specimens were obtained from 6 Quarter Horses with PSSM and 8 Quarter Horse or Thoroughbred control horses. PROCEDURES: Maximal activity of glycogenolytic and glycolytic enzymes was determined spectrophotometrically. Maximal activity of PFK was determined for each horse at pH 8.0, and at pH 7.0 when variable concentrations of the activators, fructose 6 phosphate, fructose 2,6 bisphosphate, and adenosine monophosphate or inhibitors adenosine triphosphate and citrate were added to the reaction mixture. Relative activity was calculated as activity at pH 7/maximal PFK activity. RESULTS: Deficiencies in glycogenolytic or glycolytic enzyme activities were not evident in horses with PSSM. Differences between horses with PSSM and control horses in relative activity of PFK were not apparent for any of the activators or inhibitors used in the study. CONCLUSIONS: In a group of horses with PSSM, we were unable to detect a glycogenolytic or glycolytic enzyme deficiency or abnormality in the allosteric regulation of PFK. CLINICAL RELEVANCE: Although PSSM is clinically and histologically similar to glycogenolytic/glycolytic enzyme deficiencies in human beings and other animal species, abnormalities in this metabolic pathway are not present in horses with PSSM.

Adenosine Monophosphate↗
PubMed2000

Allosterically controllable ribozymes with biosensor functions.

The concept of allosteric regulation has already been exploited in the creation of artificial ribozymes and the activities of certain ribozymes can be controlled allosterically by specific effectors. Ribozymes with such properties are in the spotlight as biosensors. Such artificial allosterically regulated ribozymes have potential utility as nucleic-acid-based biosensors.

Biosensing Techniques↗
PubMed1998

The regulation of Escherichia coli glutamine synthetase revisited: role of 2-ketoglutarate in the regulation of glutamine synthetase adenylylation state.

The regulation of Escherichia coli glutamine synthetase (GS) by reversible adenylylation has provided one of the classical paradigms for signal transduction by cyclic cascades. Yet, many mechanistic features of this regulation remain to be elucidated. We examined the regulation of GS adenylylation state in a reconstituted system containing GS, adenylyltransferase (ATase), the PII signal transduction protein that controls ATase, and the uridylyltransferase/uridylyl-removing enzyme (UTase/UR), which has a role in regulating PII. In this reconstituted bicyclic cascade system, the adenylylation state of GS was regulated reciprocally by the small molecule effectors 2-ketoglutarate and glutamine at physiological effector concentrations. By examination of the individual regulatory monocycles and comparison to the bicyclic system and existing data, we could deduce that the only sensors of 2-ketoglutarate were PII and PII-UMP. At physiological conditions, we observed that the main role of 2-ketoglutarate in bringing about the deadenylylation of GS was to inhibit GS adenylylation, and this was due to the allosteric regulation of PII activity. Glutamine acted as an allosteric regulator of both ATase and UTase/UR. We also compared the regulation of GS adenylylation state to the regulation of phosphorylation state of the transcription factor NRI (NtrC) in a reconstituted bicyclic system containing NRI, the bifunctional kinase/phosphatase NRII (NtrB), PII, and the UTase/UR. This comparison indicated that, at a fixed 2-ketoglutarate concentration, the regulation of GS adenylylation state by glutamine was sharper and occurred at a higher concentration than did the regulation of NRI phosphorylation. The possible biological implications of this regulatory arrangement are discussed.

Adenosine Triphosphate↗