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PubMed1977

[Allosteric properties of muscle creatine kinase].

The dependence of the reaction rate on substrate concentrations at pH 8.0--7.5 does not submit the Michaelis-Menten kinetics. The dependence of v on Mg-ATP is described with a curve having an intermediate plateau. The dependence of v on the creatine concentration is expressed by a curve, which is not hyperbolic. In this case the index of the substrate concentration, (q), is variable, and it increases with the increase of creatine concentration from 1 to 2 (at the presence of effectors, PEP and ADP,--from 1 to 3.5). The specific creatine kinase activity increases 3--4-fold with protein dilution, but this effect is not observed in the presence of inhibitors to FDP and PEP. Creatine kinase is desensibilized with respect to FDP and PEP after a prolonged storage. The data obtained and the presence of an effector set indicate, that muscle creatine kinase is a regulated allosteric enzyme.

Adenosine Diphosphate↗
PubMed1990

Modelling allosteric processes in E coli aspartate transcarbamylase.

The allosteric properties of aspartate transcarbamylase from E coli have been investigated by a combination of genetic, biochemical and structural studies. Based on the X-ray structures of the enzyme in T and R state established by Lipscomb et al, we have analyzed the interactions between the 12 polypeptide chains and have identified subunit interfaces that play a major part in the allosteric mechanism: the c1c4 interface between the 2 catalytic trimers, and one of 2 different interfaces between catalytic and regulatory chains, the c1r4 interface, which exists only in T state. We have modelled mutations affecting these interfaces: mutation pAR5 in the gene coding for r chains concerns the c1r4 interface, mutation Tyr----Phe 240 in the gene coding for c chains, the c1c4 interface. Both mutant proteins have reduced cooperativity and/or allosteric regulation by CTP and ATP. Molecular mechanic simulations lead to specific proposals for the structural origin of these effects, and some of the proposals can be checked by site-directed mutagenesis. Finally, we have modelled substrates bound at the active site of the T state, which binds aspartate less tightly than the R state and for which X-ray structures of bound substrate analogs were not available.

Allosteric Regulation↗
PubMed1994

Allosteric control of the substrate specificity of the anaerobic ribonucleotide reductase from Escherichia coli.

The reduction of ribonucleotides is catalyzed by different enzymes in aerobic and anaerobic Escherichia coli, each with a different primary and quaternary structure. Here, we describe the allosteric regulation of the substrate specificity of the anaerobic ribonucleoside triphosphate reductase. The enzyme reduced ribonucleotides at a low basal rate. Reduction was stimulated up to 10-fold by an appropriate modulator (dGTP for ATP reduction, ATP for CTP and UTP reduction, and dTTP for GTP reduction). dGTP and dTTP inhibited the reduction of the "incorrect" substrate; dATP inhibited reduction of all four. From kinetic, effector binding, and competition experiments we conclude that the enzyme has two classes of sites, one that binds ATP and dATP and regulates pyrimidine ribonucleotide reduction ("pyrimidine site"), the other that binds dATP, dGTP, and dTTP and regulates purine ribonucleotide reduction ("purine site"). This model differs slightly from the model for the aerobic reductase, but the physiological consequences remain the same and explain how a single enzyme can provide a balanced supply of the four dNTPs. The similarity of a highly sophisticated control mechanism for the aerobic and anaerobic enzymes suggests that both arose by divergent evolution from a common ancestor, in spite of their different structures.

Adenosine Triphosphate↗
PubMed1992

Resolution of brewer's yeast pyruvate decarboxylase into multiple isoforms with similar subunit structure and activity using high-performance liquid chromatography.

A method for the purification of brewer's yeast pyruvate decarboxylase (EC 4.1.1.1) that resolves the enzyme into multiple active isoforms was developed. Seven activity fractions are resolved by DEAE HPLC chromatography. Among these fractions, three distinct subunit composition isoforms are apparent by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: alpha 4, a homotetrameric holoenzyme consisting of the lower mass subunit; alpha 2 beta 2, a heterotetrameric holoenzyme consisting of lower and higher mass subunits; and beta 4, a homotetrameric holoenzyme consisting of the higher mass subunit. Beta 4 is a heretofore unreported form which may represent the unproteolyzed form of the enzyme. The Km and Vmax for the alpha 4 and beta 4 isoforms are identical within the limits of experimental error, as is their behavior vis-à-vis the allosteric regulator pyruvamide. All active isoforms exist as tetramers according to gel filtration analysis under native conditions. The purification has been successfully applied to pyruvate decarboxylase isolated from two different species of yeast and therefore is likely to be of general utility for purification of this enzyme from other yeast sources. Conditions under which all three isoforms demonstrate exceptional stability, making them amenable to prolonged physicochemical studies at 4 degrees C and even at room temperature are reported.

Allosteric Regulation↗
PubMed1980

The effect of the nitrosourea analog of thymidine, 3'-[3-(2-chloroethyl)-3-nitrosoureido]-3'-deoxythymidine, on Escherichia coli thymidine kinase.

Escherichia coli thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) is irreversibly inactivated by incubation with 3'-[3-(2-chloroethyl)-3-nitrosoureido]-3'-deoxythymidine (3'-CTNU). The inactivation of the enzyme followed first-order kinetics even after loss of 96% of the original activity. This indicates that the inactivation process is a one-kill phenomenon and not a generation of less active enzyme. The addition of a preincubated aqueous solution of 3'-CTNU to the enzyme reaction mixture did not inactivate the enzyme. ATP . Mg2+ but not thymidine protects the enzyme from inactivation by 3'-CTNU. The allosteric regulators, dTTP, dCTP and dCDP also afforded complete protection of the enzyme from inactivation by 3'-CTNU. These data indicate that the dimer form of the enzyme is completely resistant to inactivation by 3'-CTNU, but the monomer form of the enzyme is sensitive. The specificity of the protection is supported by the finding that neither ATP . Mg2+ nor thymidine protect yeast alcohol dehydrogenase from inactivation by this nitrosourea analog of thymidine.

Adenosine Triphosphate↗
PubMed1983

Ligand requirements for the relaxation of adenylate cyclase from activated and inhibited states.

The turkey erythrocyte adenylate cyclase system binds tightly the inhibitory nucleotide GDP, and a pretreatment step with isoproterenol and GMP is required to restore activation. Under identical pretreatment conditions, the release of labeled nucleotide is complete within 1 min whereas the restoration of activation by Gpp(NH)p requires 15 min. A study of the ligand requirements of the slow step shows the following: (a) The role of GMP is that of an obligatory allosteric regulator. (b) Cholera toxin modification of the system abolishes the requirement for GMP with a considerable enhancement in the reaction rate. (c) GMP is without effect on the relaxation process with the activator Gpp(NH)p as the resident nucleotide. In sharp contrast, ethylenediamine-tetraacetic acid (without effect in a GDP-occupied complex) markedly potentiates alterations from the Gpp(NH)p-occupied state. (d) Formation of a GDP/guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) hybrid leads to the suppression of both F- and Gpp(NH)p activation. F- activation is restored by isoproterenol alone, while GMP is still required to restore Gpp(NH)p activation. The results suggest that covalent modification or nucleotide analogue occupancy of the regulatory complex can modify the allosteric role for GMP, with consequences for the rate of the slow step.

Adenylyl Cyclases↗
PubMed2000

Purification and properties of the first-identified, archaeal, ATP-dependent 6-phosphofructokinase, an extremely thermophilic non-allosteric enzyme, from the hyperthermophile Desulfurococcus amylolyticus.

The ATP-dependent 6-phosphofructokinase (ATP-PFK) of the hyperthermophilic archaeon Desulfurococcus amylolyticus was purified 1,500-fold to homogeneity. The enzyme had an apparent molecular mass of 140 kDa and was composed of a single type of subunit of 33 kDa suggesting a homotetrameric (alpha4) structure. The N-terminal amino acid sequence did not show significant similarity to ATP-PFKs isolated from eubacteria and eukarya. Kinetic constants of the enzyme were determined for both reaction directions at pH 6 and at 85 degrees C. Rate dependence on all substrates followed Michaelis-Menten kinetics. The apparent K(m)s for ATP and fructose 6-phosphate (forward reaction) were 0.28 and 1.17 mM, respectively; the apparent V(max) was about 41 U/mg. ATP could not be replaced by pyrophosphate (PPi) or ADP as phosphoryl donor, thus defining the enzyme as an ATP-dependent PFK. In addition to ATP (100%), the enzyme accepted GTP (97%), ITP (130%), UTP (84%), CTP (55%) and, less effectively, acetyl phosphate (13%) as phosphoryl donors. Enzyme activity was not allosterically regulated by classical effectors of ATP-PFKs such as ADP, AMP, and phosphoenolpyruvate or citrate. The enzyme also catalysed in vitro the reverse reaction with an apparent K(m) for fructose-1,6-bisphosphate and ADP of 16.7 and 0.5 mM, respectively, and an apparent V(max) of about 4.5 U/mg. Divalent cations were required for maximal activity; Mg2+, which was most effective, could be replaced partially by Ni2+, Mn2+ or Co2+. The enzyme had a temperature optimum of 90 degrees C and showed a significant thermostability up to 100 degrees C, which is in accordance with its physiological function under hyperthermophilic conditions. This is the first description of an ATP-dependent PFK from the domain of archaea, characterized as an extremely thermophilic, non-allosteric enzyme.

Adenosine Triphosphate↗
PubMed1999

The vitamin D receptor binds in a transcriptionally inactive form and without a defined polarity on a retinoic acid response element.

Heterodimers of the vitamin D receptor (VDR) with the retinoid X receptor (RXR) bind in a transcriptionally unproductive manner to the retinoic acid response element present in the retinoic acid receptor-beta2 promoter. This element is composed of a direct repeat (DR) of the sequence PuGTTCA spaced by five nucleotides. However, the same sequence separated by three nucleotides (DR3) acts as a strong vitamin D response element. Here we show that the polarity of binding of the heterodimers to the DR3 was 5'-RXR-VDR-3', whereas on the DR5, both heterodimeric partners bind indistinctly to the 5' or 3' hemi-sites. These results suggest that the response elements can allosterically regulate the conformation of the receptors to determine positive or negative regulation of gene expression. Despite the altered polarity, the DR5-bound heterodimer was able to recruit the nuclear receptor coactivator ACTR in a vitamin D-dependent fashion. Furthermore, binding of the corepressor SMRT (silencing mediator of retinoid and thyroid hormone receptors) to the RXR/VDR heterodimer on a DR5 was not observed. Binding of RXR/VDR heterodimers to DRs with different transcriptional outcomes may generate selectivity and provide a greater complexity and flexibility to the vitamin D responses.

Allosteric Regulation↗
PubMed1996

Protein function in the crystal.

Protein crystals contain wide solvent-filled channels that allow for traffic of metabolites and intramolecular motility. Ligand binding, catalysis and allosteric regulation occur in the crystalline environment but intermolecular interactions may hinder function-associated transitions and alter activity with respect to solution. Lattice constraints have, however, provided the opportunity to isolate and characterize conformational states that are poorly populated in solution. New methods are being developed to initiate reactions rapidly and synchronously throughout the crystal and to monitor their time course. A model consistent with kinetics in the crystal is necessary to interpret the results of time-resolved macromolecular crystallography.

Allosteric Regulation↗
PubMed2001

Allosteric regulatory step and configuration of the ATP-binding pocket in atrial natriuretic factor receptor guanylate cyclase transduction mechanism.

The atrial natriuretic factor (ANF) signal transduction mechanism consists of the transformation of the signal information into the production of cyclic GMP. The binding of ANF to its receptor, which is also a guanylate cyclase, generates the signal. This cyclase has been termed atrial natriuretic factor receptor guanylate cyclase, ANF-RGC. ANF-RGC is a single transmembrane-spanning protein. The ANF receptor domain resides in the extracellular region of the protein, and the catalytic domain is located in the intracellular region at the C-terminus of the protein. Thus, the signal is relayed progressively from the receptor domain to the catalytic domain, where it is converted into the formation of cyclic GMP. The first transduction step is the direct binding of ATP with ANF-RGC. This causes allosteric regulation of the enzyme and primes it for the activation of its catalytic moiety. The partial structural motif of the ATP binding domain in ANF-RGC has been elucidated, and it has been named ATP regulatory module (ARM). In this presentation, we provide a brief review of the ATP-regulated transduction mechanism and the ARM model. The model depicts a configuration of the ATP-binding pocket that has been experimentally validated, and the model shows that the ATP-dependent transduction process is a two- (or more) step event. The first step involves the binding of ATP with its ARM. This partially activates the cyclase and prepares it for the subsequent steps, which are consistent with its being phosphorylated and attaining the fully activated state.

Adenosine Triphosphate↗
PubMed2003

Allosteric enhancers of A1 adenosine receptors increase receptor-G protein coupling and counteract Guanine nucleotide effects on agonist binding.

Endogenous ligands of G protein-coupled receptors bind to orthosteric sites that are topologically distinct from allosteric sites. Certain aminothiophenes such as (2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluromethyl)-phenyl]-methanone (PD81,723) and 2-amino-4,5,6,7-tetrahydro-benzo[b]thiophen-3-yl)-biphenyl-4-yl-methanone (ATL525) are positive allosteric regulators, or enhancers, of the human A1 adenosine receptor (A1AR). In equilibrium binding assays, 125I-N6-aminobenzyladenosine (125I-ABA) binds to two affinity states of A1AR with KD-high (0.33 microM) and KD-low ( approximately 10 nM). Enhancers have little effect on KD-high but convert all A1AR binding sites to the high-affinity state. Enhancers decrease the potency of guanosine 5'-O-(3-thio)triphosphate (GTPgammaS) as an inhibitor of agonist binding by 100-fold and increase agonist-stimulated guanine nucleotide exchange. The association of 125I-ABA to high-affinity receptors on Chinese hamster ovary (CHO)-hA1 membranes does not follow theoretical single-site association kinetics but is approximated by a bi-exponential equation with t1/2 values of 1.85 and 12.8 min. Allosteric enhancers selectively increase the number of slow binding sites, possibly by stabilizing newly formed receptor-G protein complexes. A new rapid assay method scores enhancer activity on a scale from 0 to 100 based on their ability to prevent the rapid dissociation of 125I-ABA from A1AR in response to GTPgammaS. Compared with PD81,723, ATL525 (100 microM) scores higher (27 versus 79) and has less antagonist activity. ATL525 functionally enhances A1 signaling to inhibit cAMP accumulation in CHO-hA1 cells. These data suggest that simultaneously binding orthosteric and allosteric enhancer ligands convert the A1AR from partly to fully coupled to G proteins and prevents rapid uncoupling upon binding of GTPgammaS.

Adenosine A1 Receptor Agonists↗
PubMed1985

Ribonucleotide reductase and deoxyribonucleotide pools.

Ribonucleotide reductase plays a crucial role in the maintenance of dNTP pools. A molecular model for the allosteric regulation of the enzyme is substantiated by the effects of nucleosides or inhibitors on pool sizes in intact cells as well as by manifestations of mutations of the reductase and other enzymes involved in deoxynucleoside metabolism. Studies of the dynamics of dNTP pools in 3T6 cells suggest compartmentation of dCTP pools, with perferential DNA synthesis from a pool labeled from cytidine. In contrast, no kinetic evidence for more than one dTTP pool was obtained. Our experiments also suggest that all dTTP formed in growing 3T6 cells is used up for DNA synthesis.

Allosteric Regulation↗
PubMed1998

Activation of thiamin diphosphate in enzymes.

Activation of the coenzyme ThDP was studied by measuring the kinetics of deprotonation at the C2 carbon of thiamin diphosphate in the enzymes pyruvate decarboxylase, transketolase, pyruvate dehydrogenase complex, pyruvate oxidase, in site-specific mutant enzymes and in enzyme complexes containing coenzyme analogues by proton/deuterium exchange detected by 1H-NMR spectroscopy. The respective deprotonation rate constant is above the catalytic constant in all enzymes investigated. The fast deprotonation requires the presence of an activator in pyruvate decarboxylase from yeast, showing the allosteric regulation of this enzyme to be accomplished by an increase in the C2-H dissociation rate of the enzyme-bound thiamin diphosphate. The data of the thiamin diphosphate analogues and of the mutant enzymes show the N1' atom and the 4'-NH2 group to be essential for the activation of the coenzyme and a conserved glutamate involved in the proton abstraction mechanism of the enzyme-bound thiamin diphosphate.

Allosteric Regulation↗
PubMed2006

The allosteric enhancer PD81,723 increases chimaeric A1/A2A adenosine receptor coupling with Gs.

PD81,723 {(2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluromethyl)-phenyl]methanone} is a selective allosteric enhancer of the G(i)-coupled A1 AR (adenosine receptor) that is without effect on G(s)-coupled A2A ARs. PD81,723 elicits a decrease in the dissociation kinetics of A1 AR agonist radioligands and an increase in functional agonist potency. In the present study, we sought to determine whether enhancer sensitivity is dependent on coupling domains or G-protein specificity of the A1 AR. Using six chimaeric A1/A2A ARs, we show that the allosteric effect of PD81,723 is maintained in a chimaera in which the predominant G-protein-coupling domain of the A1 receptor, the 3ICL (third intracellular loop), is replaced with A2A sequence. These chimaeric receptors are dually coupled with G(s) and G(i), and PD81,723 increases the potency of N6-cyclopentyladenosine to augment cAMP accumulation with or without pretreatment of cells with pertussis toxin. Thus PD81,723 has similar functional effects on chimaeric receptors with A1 transmembrane sequences that couple with G(i) or G(s). This is the first demonstration that an allosteric regulator can function in the context of a switch in G-protein-coupling specificity. There is no enhancement by PD81,723 of G(i)-coupled A2A chimaeric receptors with A1 sequence replacing A2A sequence in the 3ICL. The results suggest that the recognition site for PD81,723 is on the A1 receptor and that the enhancer acts to directly stabilize the receptor to a conformational state capable of coupling with G(i) or G(s).

Adenosine↗
PubMed2001

Structural analysis of adenylate cyclases from Trypanosoma brucei in their monomeric state.

Cyclic AMP is a major trigger of the differentiation process of Trypanosoma brucei, a bloodstream parasite causing sleeping sickness. Its generation in trypanosomes is accomplished by a unique battery of membrane-bound adenylate cyclases (ACs). We have determined the high-resolution X-ray structures of the catalytic domains of two trypanosomal ACs (tACs), GRESAG4.1 and GRESAG4.3. The tAC domains are structurally highly related to the AC domains of higher eukaryotes, but also comprise a highly conserved structural element near the active site, the Delta-subdomain. A cavity below the Delta-subdomain might correspond to an allosteric regulator site as indicated by the stereospecific binding of a single (2S,3S)-1,4- dimercapto-2,3-butanediol molecule. In three different crystal forms, the tAC domains are exclusively observed in a monomeric, catalytically inactive state. Biochemical analysis and the mutagenesis profile of GRESAG4.1 confirmed a common catalytic mechanism of tACs that involves transient dimerization of the AC domain. A low dimerization tendency might play a regulatory role in T. brucei if the activation of tACs is similarly driven by ligand-induced dimerization as in membrane-bound guanylate cyclases.

Adenylyl Cyclases↗
PubMed2004

NMR study of nucleotide-induced changes in the nucleotide binding domain of Thermus thermophilus Hsp70 chaperone DnaK: implications for the allosteric mechanism.

We present an NMR investigation of the nucleotide-dependent conformational properties of a 44-kDa nucleotide binding domain (NBD) of an Hsp70 protein. Conformational changes driven by ATP binding and hydrolysis in the N-terminal NBD are believed to allosterically regulate substrate affinity in the C-terminal substrate binding domain. Several crystal structures of Hsc70 NBDs in different nucleotide states have, however, not shown significant structural differences. We have previously reported the NMR assignments of the backbone resonances of the NBD of the bacterial Hsp70 homologue Thermus thermophilus DnaK in the ADP-bound state. In this study we show, by assigning the NBD with the ATP/transition state analogue, ADP.AlFx, bound, that it closely mimics the ATP-bound state. Chemical shift difference mapping of the two nucleotide states identified differences in a cluster of residues at the interface between subdomains 1A and 1B. Further analysis of the spectra revealed that the ATP state exhibited a single conformation, whereas the ADP state was in slow conformational exchange between a form similar to the ATP state and another state unique to the ADP-bound form. A model is proposed of the allosteric mechanism based on the nucleotide state altering the balance of a dynamic equilibrium between the open and closed states. The observed chemical shift perturbations were concentrated in an area close to a previously described J-domain binding channel, confirming the importance of that region in the allosteric mechanism.

Adenosine Diphosphate↗
PubMed2004

Volatile anesthetic modulation of oligomerization equilibria in a hexameric model peptide.

To determine if occupancy of interfacial pockets in oligomeric proteins by volatile anesthetic molecules can allosterically regulate oligomerization equilibria, variants of a three-helix bundle peptide able to form higher oligomers were studied with analytical ultracentrifugation, hydrogen exchange and modeling. Halothane shifted the oligomerization equilibria towards the oligomer only in a mutation predicted to create sufficient volume in the hexameric pocket. Other mutations at this residue, predicted to create a too small or too polar pocket, were unaffected by halothane. Inhaled anesthetic modulation of oligomerization interactions is a novel and potentially generalizable biophysical basis for some anesthetic actions.

Allosteric Regulation↗
PubMed1995

Role of residue 161 in the allosteric transitions of two bacterial phosphofructokinases.

The loop between alpha-helix 6 and beta-strand F of the phosphofructokinase (PFK) from Bacillus stearothermophilus is proposed to be important in the allosteric transition of the enzyme [Schirmer, T., & Evans, P.R. (1990) Nature 343, 140-145]. Except for residue 161, the amino acids within the loop are similar between B. stearothermophilus PFK (BsPFK) and the PFK from Escherichia coli (EcPFK). In the former enzyme, residue 161 is a glutamate, while in the latter it is a glutamine. We have used site-directed mutagenesis to investigate the importance of residue 161 for the allosteric regulation of the two enzymes by phosphoenolpyruvate (PEP), an inhibitor, and GDP, an activator. In BsPFK, glutamate 161 has been changed to a glutamine and an alanine, while in EcPFK, glutamine 161 has been changed to a glutamate, an arginine, and an alanine. The kinetic parameters of the mutant enzymes were similar to those of the respective wild types, indicating that residue 161 is not directly involved in substrate binding and catalysis. One of the EcPFK mutants, Q161A, though activated normally by GDP, was completely insensitive to PEP. This indicates that the hydrogen-bonding ability of residue 161 is critical for PEP inhibition of EcPFK and suggests that GDP activation and PEP inhibition follow different structural pathways in EcPFK. The BsPFK mutant enzymes were less sensitive to PEP inhibition and more sensitive to GDP activation, suggesting that inhibition and activation are opposed and follow a common structural pathway in agreement with a concerted allosteric mechanism.

Allosteric Regulation↗