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PubMed2000

Allosteric regulation of the class III anaerobic ribonucleotide reductase from bacteriophage T4.

Ribonucleotide reductase (RNR) is an essential enzyme in all organisms. It provides precursors for DNA synthesis by reducing all four ribonucleotides to deoxyribonucleotides. The overall activity and the substrate specificity of RNR are allosterically regulated by deoxyribonucleoside triphosphates and ATP, thereby providing balanced dNTP pools. We have characterized the allosteric regulation of the class III RNR from bacteriophage T4. Our results show that the T4 enzyme has a single type of allosteric site to which dGTP, dTTP, dATP, and ATP bind competitively. The dissociation constants are in the micromolar range, except for ATP, which has a dissociation constant in the millimolar range. ATP and dATP are positive effectors for CTP reduction, dGTP is a positive effector for ATP reduction, and dTTP is a positive effector for GTP reduction. dATP is not a general negative allosteric effector. These effects are similar to the allosteric regulation of class Ib and class II RNRs, and to the class Ia RNR of bacteriophage T4, but differ from that of the class III RNRs from the host bacterium Escherichia coli and from Lactococcus lactis. The relative rate of reduction of the four substrates was measured simultaneously in a mixed-substrate assay, which mimics the physiological situation and illustrates the interplay between the different effectors in vivo. Surprisingly, we did not observe any significant UTP reduction under the conditions used. Balancing of the pyrimidine deoxyribonucleotide pools may be achieved via the dCMP deaminase and dCMP hydroxymethylase pathways.

Adenosine Triphosphate↗
PubMed1997

The role of alpha1 and alpha6 subtype amino-terminal domains in allosteric regulation of gamma-aminobutyric acida receptors.

The gamma-aminobutyric acidA (GABA) receptor in the mammalian central nervous system is composed of pentameric combinations of alpha1-6, beta1-4, gamma1-3, delta1, and/or epsilon1 subunit subtypes. Although each of the different subunits influences the functional properties of gamma-aminobutyric acidA receptors (GABARs), the alpha subunit subtypes have been shown to be important for activation of the receptor by GABA and pentobarbital and the regulation of GABARs by numerous allosteric regulators, including benzodiazepines, furosemide, zinc, and lanthanum. However, with the exception of the benzodiazepines, the alpha subtype domain that is responsible for the action of these allosteric compounds is unknown. The alpha1 and alpha6 subtypes are among the most diverse of the alpha subunit family and confer a different responsiveness of GABARs to GABA and many of the allosteric modulators. These regulatory compounds act after extracellular application and therefore likely act on extracellular GABAR sites, the largest of which is the amino-terminal extracellular domain. To determine the role of this domain in the action of these allosteric regulatory agents, we constructed chimeras of the rat alpha1 and alpha6 subtypes with a splice site within the first putative transmembrane domain (TM). This separated the large extracellular amino-terminal domain from the transmembrane, intracellular, and TM2-3 and carboxyl-terminal extracellular domains of the subunit. The chimeric subtypes were expressed in L929 fibroblasts along with beta3 and gamma2L subtypes, and their pharmacological properties were determined with whole-cell electrophysiological recording. The alpha subtype amino-terminal extracellular domain was the primary determinant of GABA sensitivity and was responsible for the functional properties of activation by pentobarbital, sensitivity to diazepam, potentiation by lanthanum, and high affinity inhibition by furosemide. The remaining carboxyl-terminal domains influenced the GABA sensitivity and determined zinc sensitivity and low affinity inhibition by furosemide. Both domains were apparently required for inhibition by lanthanum.

Allosteric Regulation↗
PubMed1994

Heparin cofactor II is regulated allosterically and not primarily by template effects. Studies with mutant thrombins and glycosaminoglycans.

Besides its critical role in hemostasis, the serine protease thrombin also participates in wound healing, inflammation, and atherosclerosis. Thrombin is inhibited by the serpins antithrombin and heparin cofactor II (HCiI) in reactions that are accelerated markedly by specific glycosaminoglycans. Following vascular injury, thrombin must be inhibited at both intravascular and extravascular sites that impose different constraints on the recognition of thrombin by these inhibitors. The present study examines the role of anion-binding exosite II of thrombin in the interaction with glycosaminoglycans and HCII. Acceleration of thrombin inhibition by serpins in the presence of glycosaminoglycans is proposed to occur by a template mechanism, in which inhibitor and protease bind simultaneously to the same glycosaminoglycan chain, facilitating their interaction. According to the template model, disruption of protease binding to glycosaminoglycan should significantly reduce acceleration of the inhibition. Specific mutations in exosite II (R89E, R245E, K248E, and K252E) disrupted thrombin binding to both dermatan sulfate and heparin, indicating that both glycosaminoglycans bind to a common site in exosite II. The same mutations markedly decreased the rate constant for thrombin inhibition by antithrombin-heparin (up to 100-fold) but had little effect on the rate constant for thrombin inhibition by HCII-heparin (7-fold maximal reduction) and no effect on the rate constant for thrombin inhibition by HCII-dermatan sulfate. These results are incompatible with a template model for thrombin inhibition by HCII and dermatan sulfate. In the presence of glycosaminoglycan, HCII and antithrombin interact with opposing thrombin exosites and use distinct mechanisms of glycosaminoglycan catalysis. Antithrombin employs a template mechanism that requires heparin to interact with thrombin exosite II, whereas HCII employs an allosteric mechanism that requires thrombin exosite I but is largely independent of exosite II. These findings have potential implications for glycosaminoglycan therapy and for the respective physiologic roles of HCII and antithrombin.

Allosteric Regulation↗
PubMed1997

A (His)6-tagged recombinant barley (Hordeum vulgare L.) endosperm ADP-glucose pyrophosphorylase expressed in the baculovirus-insect cell system is insensitive to allosteric regulation by 3-phosphoglycerate and inorganic phosphate.

ADP-glucose pyrophosphorylase from photosynthetic tissue is allosterically regulated by 3-phosphoglycerate and inorganic phosphate. In contrast, data from our laboratory indicated that the major AGPase from barley seeds is insensitive to these regulators. Verification of this conclusion has, however, been hindered by the proteolytic degradation of the enzyme from seeds. This report characterizes the barley seed AGPase expressed in the baculovirus-insect cell system, confirming that lack of allosteric regulation by 3-PGA/Pi is an intrinsic property of the enzyme. Purification of the enzyme was by Ni2+-NTA agarose chromatography using a (His)6 tag attached to the N-terminus of the small AGPase subunit.

Allosteric Regulation↗
PubMed1994

Identification of a site necessary for allosteric regulation in T4-phage deoxycytidylate deaminase.

An allosteric inhibitor of dCMP deaminase, dTTP, forms a photolabile covalent bond with T4-phage dCMP deaminase in the presence of UV light at 254 nm. The importance of the methyl group in this process is supported by the findings that dUTP, also an allosteric inhibitor, does not photofix to the enzyme and that tritium is released from [methyl-3H dTTP during the course of the photofixation. That the bond formed is photolabile is demonstrated by the fact that tritium is released by about 10-fold over the amount of nucleotide that is photofixed. The amino acid that covalently binds dTTP in T4-dCMP deaminase was identified as Phe112. On conversion of Phe112 to an alanine by site-directed mutagenesis, there was a dramatic change in the enzyme's response to its allosteric effectors when measured early in the reaction, in that the mutant enzyme was as active as the wild-type even in the absence of dCTP and was only weakly inhibited by dTTP. However, after 10-15% of the substrate had been deaminated, the reaction rate fell off rather markedly, indicating either that an inhibitor was being accumulated on the enzyme or that the enzyme was being irreversibly inactivated with time. That the latter was not the case was shown by the addition of dCTP to the reaction, which restored the rate to that expected when it was present initially. Furthermore, we showed that, consistent with the observed loss of allosteric regulation by dCTP and dTTP, the affinity of the mutant enzyme for dTTP and dCTP as determined by binding studies was greatly reduced relative to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed2001

Proof of principle in a de novo designed protein maquette: an allosterically regulated, charge-activated conformational switch in a tetra-alpha-helix bundle.

New understanding of the engineering and allosteric regulation of natural protein conformational switches (such as those that couple chemical and ionic signals, mechanical force, and electro/chemical free energy for biochemical activation, catalysis, and motion) can be derived from simple de novo designed synthetic protein models (maquettes). We demonstrate proof of principle of both reversible switch action and allosteric regulation in a tetra-alpha-helical bundle protein composed of two identical di-helical subunits containing heme coordinated at a specific position close to the disulfide loop region. Individual bundles assume one of two switch states related by large-scale mechanical changes: a syn-topology (helices of the different subunits parallel) or anti-topology (helices antiparallel). Both the spectral properties of a coproporphyrin probe appended to the loop region and the distance-dependent redox interaction between the hemes identify the topologies. Beginning from a syn-topology, introduction of ferric heme in each subunit (either binding or redox change) shifts the topological balance by 25-50-fold (1.9-2.3 kcal/mol) to an anti-dominance. Charge repulsion between the two internal cationic ferric hemes drives the syn- to anti-switch, as demonstrated in two ways. When fixed in the syn-topology, the second ferric heme binding is 25-80-fold (1.9-2.6 kcal/mol) weaker than the first, and adjacent heme redox potentials are split by 80 mV (1.85 kcal/mol), values that energetically match the shift in topological balance. Allosteric and cooperative regulation of the switch by ionic strength exploits the shielded charge interactions between the two hemes and the exposed, cooperative interactions between the coproporphyrin carboxylates.

Allosteric Regulation↗
PubMed1995

Molecular determinants of cardiac Ca2+ channel pharmacology. Subunit requirement for the high affinity and allosteric regulation of dihydropyridine binding.

Cardiac L-type Ca2+ channels are multisubunit complexes composed of alpha 1C, alpha 2 delta, and beta 2 subunits. We tested the roles of these subunits in forming a functional complex by characterizing the effects of subunit composition on dihydropyridine binding, its allosteric regulation, and the ability of dihydropyridines to inhibit channel activity. Transfection of COS.M6 cells with cardiac alpha 1C-a (alpha 1) led to the appearance of dihydropyridine ([3H]PN200-110) binding which was increased by coexpression of cardiac beta 2a (beta), alpha 2 delta a (alpha 2), and the skeletal muscle gamma. Maximum binding was achieved when cells expressed alpha 1, beta, and alpha 2. Cells transfected with alpha 1 and beta had a binding affinity that was 5-10-fold lower than that observed in cardiac membranes. Coexpression of alpha 2 normalized this affinity. (-)-D600 and diltiazem both partially inhibited PN200-100 binding to cardiac microsomes, but stimulated binding in cells transfected with alpha 1 and beta. Again, coexpression of alpha 2 normalized this allosteric regulation. Therefore coexpression of alpha 1 beta and alpha 2 completely reconstituted high affinity dihydropyridine binding and its allosteric regulation as observed in cardiac membranes. Skeletal muscle gamma was not required for this reconstitution. Expression in Xenopus oocytes demonstrated that coexpression of alpha 2 with alpha 1 beta increased the potency and maximum extent of block of Ca2+ channel currents by nisoldipine, a dihydropyridine Ca2+ channel antagonist. Our results demonstrate that alpha 2 subunits are essential components of the cardiac L-type Ca2+ channel and predict a minimum subunit composition of alpha 1C beta 2 alpha 2 delta for this channel.

Allosteric Regulation↗
PubMed2002

Ribonucleotide reductases: the evolution of allosteric regulation.

Ribonucleotide reductases catalyze in all living organisms the production of the deoxyribonucleotides required for DNA replication and repair. Their appearance during evolution was a prerequisite for the transition from the "RNA world," where RNA sufficed for both catalysis and information transfer, to today's situation where life depends on the interplay among DNA, RNA, and protein. Three classes of ribonucleotide reductases exist today, widely differing in their primary and quaternary structures but all with a highly similar allosteric regulation of their substrate specificity. Here, I discuss the diversities between the three classes, describe their allosteric regulation, and discuss the evidence for their evolution. The appearance of oxygen on earth provided the likely driving force for enzyme diversification. From today's characteristics of the three classes, including their allosteric regulation, I propose that the anaerobic class III reductases with their iron-sulfur cluster and the requirement for S-adenosylmethionine for the generation of a glycyl protein free radical are the closest relatives to an ancestor ribonucleotide reductase.

Allosteric Regulation↗
PubMed1996

Residue 225 determines the Na(+)-induced allosteric regulation of catalytic activity in serine proteases.

Residue 225 in serine proteases is typically Pro or Tyr and specifies an important and unanticipated functional aspect of this class of enzymes. Proteases with Y225, like thrombin, are involved in highly specialized functions like blood coagulation and complement that are exclusively found in vertebrates. In these proteases, the catalytic activity is enhanced allosterically by Na+ binding. Proteases with P225, like trypsin, are typically involved in digestive functions and are also found in organisms as primitive as eubacteria. These proteases have no requirement for Na+ or other monovalent cations. The molecular origin of this physiologically important difference is remarkably simple and is revealed by a comparison of the Na+ binding loop of thrombin with the homologous region of trypsin. The carbonyl O atom of residue 224 makes a key contribution to the coordination shell of the bound Na+ in thrombin, but is oriented in a manner incompatible with Na+ binding in trypsin because of constraints imposed by P225 on the protein backbone. Pro at position 225 is therefore incompatible with Na+ binding and is a direct predictor of the lack of allosteric regulation in serine proteases. To directly test this hypothesis, we have engineered the thrombin mutant Y225P. This mutant has lost the ability to bind Na+ and behaves like the allosteric slow (Na(+)-free) form. The Na(+)-induced allosteric regulation also bears on the molecular evolution of serine proteases. A strong correlation exists between residue 225 and the codon used for the active site S195. Proteases with P225 typically use a TCN codon for S195, whereas proteases with Y225 use an AGY codon. It is proposed that serine proteases evolved from two main lineages: (i) TCN/P225 with a trypsin-like ancestor and (ii) AGY/Y225 with a thrombin-like ancestor. We predict that the Na(+)-induced allosteric regulation of catalytic activity can be introduced in the TCN/P225 lineage using the P225Y replacement.

Allosteric Regulation↗
PubMed2001

Coupling between substrate binding and allosteric regulation in ribozyme catalysis.

The contribution of substrate binding to allosteric regulation in the ribozyme catalysis has been investigated using allosteric ribozymes consisting of the hammerhead ribozyme and a flavin mononucleotide (FMN) aptamer. Kinetic parameters were measured for various lengths of the substrates with a wide range of binding energy. The maximum cleavage rate of each ribozyme was retained with the long substrates. However, the cleavage rates largely decreased by the truncation of the substrates according to loss in the free energy of substrate binding. The high sensitivity to the substrate lengths is attributed to the increase in the energetic requirement for the catalytic core folding, which is caused by the incorporation of the aptamer region. One role of FMN binding is assisting the promotion of the core folding through the stabilization of the aptamer domain. The allosteric effect is significantly expressed only when the substrate binding energy is insufficient for the core folding of the ribozyme-substrate complex. This type of allosteric interaction dominates the substrate dependency of another type of regulation. These results demonstrate that an adequate correlation between the type of regulation and the substrate binding is responsible for the effective allosteric interaction in the kinetic process.

Allosteric Regulation↗
PubMed2001

Reverse genetics of Escherichia coli glycerol kinase allosteric regulation and glucose control of glycerol utilization in vivo.

Reverse genetics is used to evaluate the roles in vivo of allosteric regulation of Escherichia coli glycerol kinase by the glucose-specific phosphocarrier of the phosphoenolpyruvate:glycose phosphotransferase system, IIA(Glc) (formerly known as III(glc)), and by fructose 1,6-bisphosphate. Roles have been postulated for these allosteric effectors in glucose control of both glycerol utilization and expression of the glpK gene. Genetics methods based on homologous recombination are used to place glpK alleles with known specific mutations into the chromosomal context of the glpK gene in three different genetic backgrounds. The alleles encode glycerol kinases with normal catalytic properties and specific alterations of allosteric regulatory properties, as determined by in vitro characterization of the purified enzymes. The E. coli strains with these alleles display the glycerol kinase regulatory phenotypes that are expected on the basis of the in vitro characterizations. Strains with different glpR alleles are used to assess the relationships between allosteric regulation of glycerol kinase and specific repression in glucose control of the expression of the glpK gene. Results of these studies show that glucose control of glycerol utilization and glycerol kinase expression is not affected by the loss of IIA(Glc) inhibition of glycerol kinase. In contrast, fructose 1,6-bisphosphate inhibition of glycerol kinase is the dominant allosteric control mechanism, and glucose is unable to control glycerol utilization in its absence. Specific repression is not required for glucose control of glycerol utilization, and the relative roles of various mechanisms for glucose control (catabolite repression, specific repression, and inducer exclusion) are different for glycerol utilization than for lactose utilization.

Allosteric Regulation↗
PubMed1997

Topology of allosteric regulation of lactose permease.

Sugar transport by some permeases in Escherichia coli is allosterically regulated by the phosphorylation state of the intracellular regulatory protein, enzyme IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system. A sensitive radiochemical assay for the interaction of enzyme IIAglc with membrane-associated lactose permease was used to characterize the binding reaction. The binding is stimulated by transportable substrates such as lactose, melibiose, and raffinose, but not by sugars that are not transported (maltose and sucrose). Treatment of lactose permease with N-ethylmaleimide, which blocks ligand binding and transport by alkylating Cys-148, also blocks enzyme IIAglc binding. Preincubation with the substrate analog beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside protects both lactose transport and enzyme IIAglc binding against inhibition by N-ethylmaleimide. A collection of lactose permease replacement mutants at Cys-148 showed, with the exception of C148V, a good correlation of relative transport activity and enzyme IIAglc binding. The nature of the interaction of enzyme IIAglc with the cytoplasmic face of lactose permease was explored. The N- and C-termini, as well as five hydrophilic loops in the permease, are exposed on the cytoplasmic surface of the membrane and it has been proposed that the central cytoplasmic loop of lactose permease is the major determinant for interaction with enzyme IIAglc. Lactose permease mutants with polyhistidine insertions in cytoplasmic loops IV/V and VI/VII and periplasmic loop VII/VIII retain transport activity and therefore substrate binding, but do not bind enzyme IIAglc, indicating that these regions of lactose permease may be involved in recognition of enzyme IIAglc. Taken together, these results suggest that interaction of lactose permease with substrate promotes a conformational change that brings several cytoplasmic loops into an arrangement optimal for interaction with the regulatory protein, enzyme IIAglc. A topological map of the proposed interaction is presented.

Allosteric Regulation↗
PubMed1991

Allosteric regulation of purine nucleoside phosphorylase.

Purine nucleoside phosphorylase (EC 2.4.2.1) from bovine spleen is allosterically regulated. With the substrate inosine the enzyme displayed complex kinetics: positive cooperativity vs inosine when this substrate was close to physiological concentrations, negative cooperativity at inosine concentrations greater than 60 microM, and substrate inhibition at inosine greater than 1 mM. No cooperativity was observed with the alternative substrate, guanosine. The activity of purine nucleoside phosphorylase toward the substrate inosine was sensitive to the presence of reducing thiols; oxidation caused a loss of cooperativity toward inosine, as well as a 10-fold decreased affinity for inosine. The enzyme also displayed negative cooperativity toward phosphate at physiological concentrations of Pi, but oxidation had no effect on either the affinity or cooperativity toward phosphate. The importance of reduced cysteines on the enzyme is thus specific for binding of the nucleoside substrate. The enzyme was modestly inhibited by the pyrimidine nucleotides CTP (Ki = 118 microM) and UTP (Ki = 164 microM), but showed greater sensitivity to 5-phosphoribosyl-1-pyrophosphate (Ki = 5.2 microM).

Allosteric Regulation↗
PubMed1998

The allosteric regulation of pyruvate kinase by fructose-1,6-bisphosphate.

BACKGROUND: Yeast pyruvate kinase (PK) catalyzes the final step in glycolysis. The enzyme therefore represents an important control point and is allosterically activated by fructose-1,6-bisphosphate (FBP). In mammals the enzyme is found as four different isozymes with different regulatory properties: two of these isozymes are produced by alternate splicing. The allosteric regulation of PK is directly related to proliferation of certain cell types, as demonstrated by the expression of an allosterically regulated isozyme in tumor cells. A model for the allosteric transition from the inactive (T) state to the active (R) state has been proposed previously, but until now the FBP-binding site had not been identified. RESULTS: We report here the structures of PK from yeast complexed with a substrate analog and catalytic metal ions in the presence and absence of bound FBP. The allosteric site is located 40 A from the active site and is entirely located in the enzyme regulatory (C) domain. A phosphate-binding site for the allosteric activator is created by residues encoded by a region of the gene corresponding to the alternately spliced exon of mammalian isozymes. FBP activation appears to induce several conformational changes among active-site sidechains through a mechanism that is most likely to involve significant domain motions, as previously hypothesized. CONCLUSIONS: The structure and location of the allosteric activator site agrees with the pattern of alternate genetic splicing of the PK gene in multicellular eukaryotes that distinguishes between a non-regulated isozyme and the regulated fetal isozymes. The conformational differences observed between the active sites of inactive and fully active PK enzymes is in agreement with the recently determined thermodynamic mechanism of allosteric activation through a 'metal relay' that increases the affinity of the enzyme for its natural phosphoenolpyruvate substrate.

Allosteric Regulation↗
PubMed1998

Mechanism of allosteric regulation of the rod cGMP phosphodiesterase activity by the helical domain of transducin alpha subunit.

The G protein alpha subunit (Galpha) is composed of two distinct folding domains: a GTP-binding Ras-like domain and an alpha helical domain (HD). We have recently reported that the helical domain (HDt) of the vertebrate visual transducin alpha subunit (Galphat) synergizes activation of retinal cyclic GMP phosphodiesterase (PDE) by activated Galphat (Liu, W., and Northup, J. K., (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12878-12883). Here, we examine the molecular basis for this HD-based signaling regulation, and we provide a new model for the activation of the target effector. The HD proteins derived from visual transducin or taste gustducin alpha subunits, but no other Galpha HD proteins, each attenuate the PDE catalytic core (Palphabeta) and synergize Galphat stimulation of the holoPDE (Palphabetagamma2) with similar apparent affinities. The data from studies of both HDt-mediated attenuation and stimulation indicate that the HDt and the PDE inhibitory subunit (Pgamma) interact with PDE at independent sites and that Palphabeta contains the binding sites for HD. The saturation of both processes by HDt displays positive cooperativity with Hill coefficients of 1.5 for the attenuation of Palphabeta activity and 2.1 for synergism of holoPDE activation. Our data suggest the that Galphat-HDt regulates PDE by allosterically decreasing the affinity of Palphabeta for Pgamma and thus simultaneously facilitating the interaction of the activated Galphat-Ras-like domain with Pgamma. Thus, we propose a new model for the high efficiency of PDE activation as well as deactivation, and, overall, a novel mechanism for controlling fidelity, sensitivity, and efficacy of G protein signaling.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1998

Allosteric regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors by thiocyanate and cyclothiazide at a common modulatory site distinct from that of 2,3-benzodiazepines.

Allosteric regulators of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors include 2,3-benzodiazepines such as GYKI 52466 and GYKI 53655 and the chaotropic anion thiocyanate that inhibit, and benzothiadiazines such as cyclothiazide that potentiate AMPA receptor currents. Here we sought to determine whether the allosteric regulators modulate AMPA receptors at a common or distinct allosteric sites by comparing their actions on AMPA- and kainate-evoked currents in cultured rat hippocampal neurons and Xenopus oocytes expressing recombinant AMPA receptor subunits. GYKI 52466 and thiocyanate blocked AMPA-evoked currents in a concentration-dependent manner (IC50 values, 8.2 microM and 1.1 mM, respectively); in contrast, kainate-evoked currents were blocked by GYKI 52466, but were potentiated by high concentrations of thiocyanate (> or = 3 mM). Thiocyanate enhanced the rate of desensitization and slowed recovery from desensitization of AMPA-evoked currents, whereas GYKI 52466 failed to affect desensitization. Among neurons in the hippocampal cultures, there was cell-to-cell variability in the sensitivity to block of AMPA-evoked currents by thiocyanate that was correlated with the degree of potentiation by cyclothiazide. Moreover, cyclothiazide caused a parallel rightward shift in the concentration-response curve for thiocyanate block, and slowed the onset of thiocyanate block to a rate that was similar to that of cyclothiazide dissociation. Together, these observations suggest that thiocyanate and cyclothiazide act at non-distinct allosteric sites. GYKI 52466 blocked AMPA receptor responses to a similar extent, irrespective of the degree of cyclothiazide potentiation. Moreover, the kinetics of GYKI 53655 block in the presence of cyclothiazide were not consistent with a competitive interaction. As is the case for cyclothiazide, SCN- exhibited greater affinity for flip than for flop AMPA receptor splice variants. In particular, GluR1flip/GluR2flip was especially sensitive to thiocyanate block. We conclude that thiocyanate, a flip-preferring allosteric modulator like cyclothiazide, appears to act by enhancing desensitization at a site that may overlap the site where cyclothiazide reduces desensitization, whereas 2,3-benzodiazepines act at a distinct site and the block does not involve a modification of desensitization.

Allosteric Regulation↗
PubMed2001

Crystal structure of ATP sulfurylase from Penicillium chrysogenum: insights into the allosteric regulation of sulfate assimilation.

ATP sulfurylase from Penicillium chrysogenum is an allosterically regulated enzyme composed of six identical 63.7 kDa subunits (573 residues). The C-terminal allosteric domain of each subunit is homologous to APS kinase. In the presence of APS, the enzyme crystallized in the orthorhombic space group (I222) with unit cell parameters of a = 135.7 A, b = 162.1 A, and c = 273.0 A. The X-ray structure at 2.8 A resolution established that the hexameric enzyme is a dimer of triads in the shape of an oblate ellipsoid 140 A diameter x 70 A. Each subunit is divided into a discreet N-terminal domain, a central catalytic domain, and a C-terminal allosteric domain. Two molecules of APS bound per subunit clearly identify the catalytic and allosteric domains. The sequence 197QXRN200 is largely responsible for anchoring the phosphosulfate group of APS at the active site of the catalytic domain. The specificity of the catalytic site for adenine nucleotides is established by specific hydrogen bonds to the protein main chain. APS was bound to the allosteric site through sequence-specific interactions with amino acid side chains that are conserved in true APS kinase. Within a given triad, the allosteric domain of one subunit interacts with the catalytic domain of another. There are also allosteric-allosteric, allosteric-N-terminal, and catalytic-catalytic domain interactions across the triad interface. The overall interactions-each subunit with four others-provide stability to the hexamer as well as a way to propagate a concerted allosteric transition. The structure presented here is believed to be the R state. A solvent channel, 15-70 A wide exists along the 3-fold axis, but substrates have access to the catalytic site only from the external medium. On the other hand, a surface "trench" links each catalytic site in one triad with an allosteric site in the other triad. This trench may be a vestigial feature of a bifunctional ("PAPS synthetase") ancestor of fungal ATP sulfurylase.

Adenosine Phosphosulfate↗
PubMed2005

Mechanism of allosteric regulation of Dnmt1's processivity.

We have analyzed the relationship between the allosteric regulation and processive catalysis of DNA methyltransferase 1 (Dnmt1). Processivity is described quantitatively in terms of turnover rate, DNA dissociation rate, and processivity probability. Our results provide further evidence that the active site and the allosteric sites on Dnmt1 can bind DNA independently. Dnmt1's processive catalysis on unmethylated DNA is partially inhibited when the allosteric site binds unmethylated DNA and fully inhibited when the allosteric site binds a single-stranded oligonucleotide inhibitor. The partial inhibition by unmethylated DNA is caused by a decrease in the turnover rate and an increase in the substrate DNA dissociation rate. Processive catalysis with premethylated DNA is not affected if the allosteric site is exposed to premethylated DNA but is fully inhibited if the allosteric site binds unmethylated DNA or poly(dA-dT). In sum, the occupancy of the allosteric site modulates the enzyme's commitment to catalysis, which reflects the nature of the substrate and the DNA bound at the allosteric site. Our in vitro results are consistent with the possibility that the processive action of Dnmt1 may be regulated in vivo by specific regulatory nucleic acids such as DNA, RNA, or poly(ADP-ribose).

Allosteric Regulation↗