Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex

The tryptophan synthase α₂β₂ bi-enzyme complex catalyzes the last two steps in the synthesis of l-tryptophan (l-Trp). The α-subunit catalyzes cleavage of 3-indole-d-glycerol 3′-phosphate (IGP) to give indole and d-glyceraldehyde 3′-phosphate (G3P). Indole is then transferred (channeled) via an interconnecting 25 Å-long tunnel, from the α-subunit to the β-subunit where it reacts with l-Ser in a pyridoxal 5′-phosphate-dependent reaction to give l-Trp and a water molecule. The efficient utilization of IGP and l-Ser by tryptophan synthase to synthesize l-Trp utilizes a system of allosteric interactions that (1) function to switch the α-site on and off at different stages of the β-subunit catalytic cycle, and (2) prevent the escape of the channeled intermediate, indole, from the confines of the α- and β-catalytic sites and the interconnecting tunnel. This review discusses in detail the chemical origins of the allosteric interactions responsible both for switching the α-site on and off, and for triggering the conformational changes between open and closed states which prevent the escape of indole from the bienzyme complex.     

Synthesis and characterization of allosteric probes of substrate channeling in the tryptophan synthase bienzyme complex

Allosteric interactions regulate substrate channeling in Salmonella typhimurium tryptophan synthase. The channeling of indole between the alpha- and beta-sites via the interconnecting 25 A tunnel is regulated by allosteric signaling arising from binding of ligand to the alpha-site, and covalent reaction of l-Ser at the beta-site. This signaling switches the alpha- and beta-subunits between open conformations of low activity and closed conformations of high activity. Our objective is to synthesize and characterize new classes of alpha-site ligands (ASLs) that mimic the binding of substrates, 3-indole-d-glycerol 3'-phosphate (IGP) or d-glyceraldehyde 3-phosphate (G3P), for use in the investigation of alpha-site-beta-site interactions. The new synthesized IGP analogues contain an aryl group linked to an O-phosphoethanolamine moiety through amide, sulfonamide, or thiourea groups. The G3P analogue, thiophosphoglycolohydroxamate, contains a hydroxamic acid group linked to a thiophosphate moiety. Crystal structures of the internal aldimine complexed with G3P and with three of the new ASLs are presented. These structural and solution studies of the ASL complexes with the internal aldimine form of the enzyme establish the following. (1) ASL binding occurs with high specificity and relatively high affinities at the alpha-site. (2) Binding of the new ASLs slows the entry of indole analogues into the beta-site by blocking the tunnel opening at the alpha-site. (3) ASL binding stabilizes the closed conformations of the beta-subunit for the alpha-aminoacrylate and quinonoid forms of the enzyme. (4) The new ASLs exhibit allosteric properties that parallel the behaviors of IGP and G3P.     

Serine modulates substrate channeling in tryptophan synthase. A novel intersubunit triggering mechanism

Tryptophan synthase, an alpha 2 beta 2 complex, is a classic example of an enzyme that is thought to "channel" a metabolic intermediate (indole) from the active site of the alpha subunit to the active site of the beta subunit. We now examine the kinetics of substrate channeling by tryptophan synthase directly by chemical quench-flow and stopped-flow methods. The conversion of indole-3-glycerol phosphate (IGP) to tryptophan at the active site proceeds at a rate of 24 s-1, which is limited by the rate of cleavage of IGP to produce indole (alpha reaction). In a single turnover experiment monitoring the conversion of radiolabeled IGP to tryptophan, only a trace of indole is detectable (less than or equal to 1% of the IGP), implying that the reaction of indole to form tryptophan must be quite fast (greater than or equal to 1000 s-1). The rate of reaction of indole from solution is much too slow (40 s-1 under identical conditions) to account for the negligible accumulation of indole in a single turnover. Therefore, the indole produced at the alpha site must be rapidly channeled to the beta site, where it reacts with serine to form tryptophan: channeling and the reaction of indole to form tryptophan must each occur at rates greater than or equal to 1000 s-1. Steady-state turnover is limited by the slow rate of tryptophan release (8 s-1). In the absence of serine, the cleavage of IGP to indole is limited by a change in protein conformation at a rate of 0.16 s-1. When the alpha beta reaction is initiated by mixing enzyme with IGP and serine simultaneously, there is a lag in the cleavage IGP and formation of tryptophan. The kinetics of the lag correspond to the rate of formation of the aminoacrylate in the reaction of serine with pyridoxal phosphate at the beta site, measured by stopped-flow methods (45 s-1). There is also a change in protein fluorescence, suggestive of a change in protein conformation, occurring at the same rate. Substitution of cysteine for serine leads to a longer lag in the kinetics of IGP cleavage and a correspondingly slower rate of formation of the aminoacrylate (6 s-1). Thus, the reaction of serine at the beta site modulates the alpha reaction such that the formation of the aminoacrylate leads to a change in protein conformation that is transmitted to the alpha site to enhance the rate of IGP cleavage 150-fold.(ABSTRACT TRUNCATED AT 400 WORDS)     

Polyphosphoinositides are derived from ether-linked inositol glycerophospholipids in rat brain

Membrane inositol glycerophospholipid (IGP) is metabolized to phosphatidylinositol-4-phosphate (PIP), phosphatidylinositol-4, 5-bisphosphate (PIP2), and inositol triphosphate (IP3) in signaling transduction. This study was carried out to determine the subclasses of IGP involved in signaling pathway. The acyl chain moieties of the phospholipids are easily modulated by dietary fatty acids. We analyzed acyl chain composition of IGP 3-subclasses, PIP and PIP2 from rat brain after feeding sunflower seed oil enriched with linoleic acid or fish oil high in eicosapentaenoic acid and docosahexaenoic acid. Long chain polyunsaturated fatty acids (LCPUFA) as eicosapentaenoic acid and docosahexaenoic acid were not incorporated into ether-linked IGP (alkenylacylglycerophosphoinositol and alkylacyl-glycerophosphoinositol), PIP and PIP2, while diacyl-glycerophosphoinositol (GPI) contained high LCPUFA. These results suggest that PIP might be phosphorylated from only the ether-linked IGP (alkenylacyl- and alkylacyl species) but not from diacyl subclass for signals to intracellular responses in the plasma membrane of rat brain.     

Allosteric effects acting over a distance of 20-25 A in the Escherichia coli tryptophan synthase bienzyme complex increase ligand affinity and cause redistribution of covalent intermediates

The reactions of L-histidine (L-His) and L-tryptophan (L-Trp) with the alpha 2 beta 2 complex of Escherichia coli tryptophan synthase are introduced as probes both of beta-subunit catalysis and of ligand-mediated alpha-beta allosteric interactions. Binding of DL-alpha-glycerol 3-phosphate (GP), an analogue of 3-indole-D-glycerol 3'-phosphate (IGP), to the alpha-catalytic site increases the affinity of alpha 2 beta 2 for L-His 4.5-fold and the affinity for L-Trp 17-fold and brings about a redistribution of beta-bound intermediates that favors the quinonoids derived from each amino acid. Inorganic phosphate (Pi) (presumably via binding to the alpha-catalytic site) influences the distribution of L-His intermediates as does GP. Previous binding studies [Heyn, M. P., & Weischet, W. O. (1975) Biochemistry 14, 2962-2968] indicate that when the phosphoryl group subsite of the alpha-catalytic site is occupied by GP or Pi, a high-affinity indole subsite is induced at the alpha-catalytic site. Interaction of benzimidazole (BZ), an analogue of indole, with this site also shifts the distribution of beta-bound L-His intermediates in favor of the L-His quinonoid. In the absence of Pi or GP, BZ interacts primarily at the beta-catalytic site and competes with L-His for the beta-subunit indole subsite. Since L-His and GP (or Pi) are substrate analogues and L-Trp is the physiological product, these allosteric effects likely take place with the natural substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arabidopsis indole synthase, a homolog of tryptophan synthase alpha, is an enzyme involved in the Trp-independent indole-containing metabolite biosynthesis

The plant tryptophan (Trp) biosynthetic pathway produces many secondary metabolites with diverse functions.Indole-3-acetic acid (IAA),proposed as a derivative from Trp or its precursors,plays an essential role in plant growth and development.Although the Trp-dependant and Trp-independent IAA biosynthetic pathways have been proposed,the enzymes,reactions and regulatory mechanisms are largely unknown.In Arabidopsis,indole-3-glycerol phosphate (IGP) is suggested to serve as a branchpoint component in the Trp-independent IAA biosynthesis.To address whether other enzymes in addition to Trp synthase α(TSA1) catalyze IGP cleavage,we identified and characterized an indole synthase (INS) gene,a homolog of TSA1 in Arabidopsis.INS exhibits different subcellular localization from TSA1 owing to the lack of chloroplast transit peptide (cTP).In silico data show that the expression levels of INS and TSA1 in all examined organs are quite different.Histochemical staining of INS promoter-GUS transgenic lines indicates that INS is expressed in vascular tissue of cotyledons,hypocotyls,roots and rosette leaves as well as in flowers and siliques.INS is capable of complementing the Trp auxotrophy of Escherichia coil △trpA strain,which is defective in Trp synthesis due to the deletion of TSA.This implies that INS catalyzes the conversion of IGP to indole and may be involved in the biosynthesis of Trp-independent IAA or other secondary metabolites in Arabidopsis.     

Crystal structure of wild-type tryptophan synthase complexed with the natural substrate indole-3-glycerol phosphate

We used freeze trapping to stabilize the Michaelis complex of wild-type tryptophan synthase and the alpha-subunit substrate indole-3-glycerol phosphate (IGP) and determined its structure to 1. 8 A resolution. In addition, we determined the 1.4 A resolution structure of the complex with indole-3-propanole phosphate (IPP), a noncleavable IGP analogue. The interaction of the 3'-hydroxyl of IGP with the catalytic alphaGlu49 leads to a twisting of the propane chain and to a repositioning of the indole ring compared to IPP. Concomitantly, the catalytic alphaAsp60 rotates resulting in a translocation of the COMM domain [betaGly102-betaGly189, for definition see Schneider et al. (1998) Biochemistry 37, 5394-5406] in a direction opposite to the one in the IPP complex. This results in loss of the allosteric sodium ion bound at the beta-subunit and an opening of the beta-active site, thereby making the cofactor pyridoxal 5'-phosphate (PLP) accessible to solvent and thus serine binding. These findings form the structural basis for the information transfer from the alpha- to the beta-subunit and may explain the affinity increase of the beta-active site for serine upon IGP binding.     

Distinct conformational dynamics and allosteric networks in alpha tryptophan synthase during active catalysis

Experimental observations of enzymes under active turnover conditions have brought new insight into the role of protein motions and allosteric networks in catalysis. Many of these studies characterize enzymes under dynamic chemical equilibrium conditions, in which the enzyme is actively catalyzing both the forward and reverse reactions during data acquisition. We have previously analyzed conformational dynamics and allosteric networks of the alpha subunit of tryptophan synthase under such conditions using NMR. We have proposed that this working state represents a four to one ratio of the enzyme bound with the indole‐3‐glycerol phosphate substrate (E:IGP) to the enzyme bound with the products indole and glyceraldehyde‐3‐phosphate (E:indole:G3P). Here, we analyze the inactive D60N variant to deconvolute the contributions of the substrate‐ and products‐bound states to the working state. While the D60N substitution itself induces small structural and dynamic changes, the D60N E:IGP and E:indole:G3P states cannot entirely account for the conformational dynamics and allosteric networks present in the working state. The act of chemical bond breakage and/or formation, or possibly the generation of an intermediate, may alter the structure and dynamics present in the working state. As the enzyme transitions from the substrate‐bound to the products‐bound state, millisecond conformational exchange processes are quenched and new allosteric connections are made between the alpha active site and the surface which interfaces with the beta subunit. The structural ordering of the enzyme and these new allosteric connections may be important in coordinating the channeling of the indole product into the beta subunit.     

Beta-elimination of indole from L-tryptophan catalyzed by bacterial tryptophan synthase: a comparison between reactions catalyzed by tryptophanase and tryptophan synthase

Although tryptophan synthase catalyzes a number of pyridoxal phosphate dependent beta-elimination and beta-replacement reactions that are also catalyzed by tryptophanase, a principal and puzzling difference between the two enzymes lies in the apparent inability of tryptophan synthase to catalyze beta-elimination of indole from L-tryptophan. We now demonstrate for the first time that the beta 2 subunit and the alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli and from Salmonella typhimurium do catalyze a slow beta-elimination reaction with L-tryptophan to produce indole, pyruvate, and ammonia. The rate of the reaction is about 10-fold higher in the presence of the alpha subunit. The rate of indole production is increased about 4-fold when the aminoacrylate produced is converted to S-(hydroxyethyl)-L-cysteine by a coupled beta-replacement reaction with beta-mercaptoethanol. The rate of L-tryptophan cleavage is also increased when the indole produced is removed by extraction with toluene or by condensation with D-glyceraldehyde 3-phosphate to form indole-3-glycerol phosphate in a reaction catalyzed by the alpha subunit of tryptophan synthase. The amount of L-tryptophan cleavage is greatest in the presence of both beta-mercaptoethanol and D-glyceraldehyde 3-phosphate, which cause the removal of both products of cleavage. The cleavage reaction is not due to contaminating tryptophanase since the activity is not inhibited by (3R)-2,3-dihydro-L-tryptophan, a specific inhibitor of tryptophanase, but is inhibited by (3S)-2,3-dihydro-L-tryptophan, a specific inhibitor of tryptophan synthase. The cleavage reaction is also inhibited by D-tryptophan, the product of a slow racemization reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circular dichroism and fluorescence studies on the binding of ligands to the alpha subunit of tryptophan synthase.

Binding to the alpha subunit of tryptophan synthase induces extrinsic Cotton effects in the substrates indole (IND), indoleglycerol phosphate (IGP), and D-glyceraldehyde-3-P (D-GAP) and in the inhibitor indolepropanol phosphate (IPP). These effects disappear when the enzyme is denatured in guanidinium chloride. The induced circular dichroism (CD) was used to determine the dissociation constant and the number of binding sites for IPP. The dissociation constant so determined is equal to 48 muM and is in good agreement with the value of 48 muM obtained by equilibrium dialysis. From the temperature dependence of the dissociation constant, a value of -2.8 kcal/mol for the binding enthalpy was obtained. The determination of dissociation constants by means of extrinsic Cotton effects is shown to be quite feasible. CD competition experiments with glycerol phosphate (GP) suggest that IPP binds bifunctionally to the enzyme: via its indole part and its phosphate group. Indolepropanol, which lacks the phosphate group, does not show an extrinsic Cotton effect. Since the induced CD is strongly dependent on the binding geometry, the close similarity between the induced spectra in IPP and IGP is additional evidence that IPP is a good substrate analog. Binding to the enzyme results in a blue shift of the IPP fluorescence emission maximum. The dissociation constant determined by fluorescence titration equals 46 muM and agrees well with the values determined by the other two methods. Previous biochemical and fast kinetic studies suggested the existence of multiple conformational states for the enzyme and of ligand-induced conformational changes. No evidence was found in the far-uv CD spectra for conformational changes upon binding of IND and D-GAP. For IPP a very small effect was observed.     

Steady-state kinetics of indole-3-glycerol phosphate synthase from Mycobacterium tuberculosis

Indole-3-glycerol phosphate synthase (IGPS) catalyzes the irreversible ring closure of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP), through decarboxylation and dehydration steps, releasing indole-3-glycerol phosphate (IGP), the fourth step in the biosynthesis of tryptophan. This pathway is essential for Mycobacterium tuberculosis virulence. Here we describe the cloning, expression, purification, and kinetic characterization of IGPS from M. tuberculosis. To perform kinetic studies, CdRP was chemically synthesized, purified, and spectroscopically and spectrometrically characterized. CdRP fluorescence was pH-dependent, probably owing to excited-state intramolecular proton transfer. The activation energy was calculated, and solvent isotope effects and proton inventory studies were performed. pH-rate profiles were carried out to probe for acid/base catalysis, showing that a deprotonated residue is necessary for CdRP binding and conversion to IGP. A model to describe a steady-state kinetic sequence for MtIGPS-catalized chemical reaction is proposed.     

Photoaffinity labeling of the indole sites on the Escherichia coli tryptophan synthase alpha-subunit

The alpha subunit of the Escherichia coli tryptophan synthase catalyzes the reversible aldolytic reaction: Indole-3-glycerol phosphate in equilibrium indole + glyceraldehyde 3-phosphate. The use of 5-azidoindole as a photoaffinity label has made the generation of a number of enzyme-substrate complexes possible, each with a given degree of saturation of the two postulated indole sites. When assayed in the reverse reaction (indole-3-glycerol phosphate synthesis), samples of alpha subunit treated at concentrations of 5-azidoindole less than or equal to 2 mM show a progressive 30-40% activation. A gradual inactivation occurs only in samples irradiated at concentrations in excess of 2 mM 5-azidoindole, and this inactivation is complete at 8-10 mM. A quantitatively similar activation occurs in the forward reaction (indole synthesis), however inactivation in this case is incomplete, with complexes treated at 8-12 mM 5-azidoindole retaining 30-40% relative activity in this reaction. When treated alpha subunits were assayed for their abilities to complement the beta 2-subunit in the reactions indole + L-serine leads to L-tryptophan + H2O and indole-3-glycerol phosphate + L-serine leads to L-tryptophan + glyceraldehyde 3-phosphate, quantitatively lesser amounts of activation followed by total inactivation are observed over a similar range of 5-azidoindole concentrations.     

Conformational changes and subunit communication in tryptophan synthase: effect of substrates and substrate analogs.

The transmission of regulatory signals between the alpha- and beta-subunits of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium has been investigated by monitoring the luminescence properties of the enzyme in the presence and in the absence of the alpha-subunit ligand DL-alpha-glycerol 3-phosphate, the alpha- and beta-subunit substrate indole, and the beta-subunit substrate analog L-histidine. The beta-subunit contains as intrinsic probes Trp-177 and pyridoxal 5'-phosphate, whereas the alpha-subunit has been mutagenized by replacing Ala-129 with a Trp residue. In contrast to the inertness of L-histidine, DL-alpha-glycerol 3-phosphate was found (i) to alter the phosphorescence spectrum of Trp-129, (ii) to shift the fluorescence thermal quenching profile of both Trp-177 and coenzyme to higher temperature, (iii) to slow down the triplet decay kinetics of Trp-177 in fluid solution, and (iv) to affect the equilibrium between different conformations of the enzyme. These findings provide direct evidence that DL-alpha-glycerol 3-phosphate binding affects the structure of the alpha-subunit and, in the presence of coenzyme, induces a conformational change in the beta-subunit that leads to a considerably more rigid structure. As opposed to DL-alpha-glycerol 3-phosphate, the shortening of the phosphorescence lifetime upon indole binding suggests that this substrate increases structural fluctuations in the beta-subunit. Implications for the mechanism of the allosteric regulation between alpha- and beta-subunits are discussed.     

Physiological and enzymatic aspects of histidine-mediated control of the tryptophan pathway

Summary It would thus appear that in Saccharomyces cerevisiae there are two forms of histidine-mediated control on the tryptophan pathway. In some strains histidine increases anthranilate synthetase and indole glycerol phosphate synthetase activities, while tryptophan synthetase decreases. In other strains histidine affects coordinately all enzymatic activities involved in tryptophan biosynthesis. The two groups of strains also differ in the formation, during the growth of the enzymatic activities involved in tryptophan biosynthesis. This difference in the relative rates at which the two enzymes are formed may explain the accumulation of intermediates in the cultural media of some strains. The derepression of anthranilate synthetase and indole glycerol phosphate synthetase activities by histidine is particularly manifest in the auxotrophic his₃ strains that show these activities very depressed in histidine starvation; large amounts of this amino acid stimulate them to a considerably greater extent than in prototrophic strains.Abbreviations IGP imidazole glycerol phosphate - InGP indole glycerol phosphate - ASase anthranilate synthetase - InGPase indole-3-glycerol phosphate synthetase - TSase tryptophan synthetase - Tris tris (hydroxymethyl)-aminomethane This investigation was supported by a research grant of C.N.R. (Consiglio Nazionale delle Ricerche, Roma).     

On the mechanism of action of Escherichia coli tryptophan synthase. Steady-state investigations.

Tryptophan synthase from Escherichia coli (L-serine hydro-lyase (adding indole), EC 4.2.1.20) synthesizes L-trypotophan from indoleglycerol phosphate and L-serine, releasing glyceraldehyde 3-phosphate, or from indole and L-serine. The latter reaction (B reaction), catalyzed either by the beta2 species or by the (alpha2 beta2) complex, has been studied by steady-state methods. A sequential mechanism is indicated. Inhibition experiments with the substrate analogue benzimidazole were carried out in order to distinguish between random and ordered mechanisms. The results are compatible with a random sequential mechanism. The dissociation constants of the enzyme-substrate complexes are evaluated. When catalyzed by the tetrameric complex (alpha2 beta2) the B reaction is inhibited by higher concentrations of the substrate indole. This inhibition does not follow the usual substrate inhibition pattern. The question whether the binding of indole to the alpha-subunit exerts an inhibitory effect on the beta2 species, possibly by reversing the activation by the alpha subunit of the beta2 species, is discussed.     

Affinities of phosphorylated substrates for the E. coli tryptophan synthase α-subunit: Roles of Ser-235 and helix-8′ dipole

The roles of Ser-235 and helix-8′ (residues 235–242) in the functional binding and turnover of phosphorylated substrates by the α-subunit of the E. coli tryptophan synthase (TSase) α₂β₂-holoenzyme complex are examined. Previous crystallographic analyses indicated that this region was one of several near the phosphate moiety of the physiological substrate, indole-3-glycerol phosphate (IGP). The peptidyl amido group of Ser-235 was suggested to H-bond to the phosphate group; a helix macrodipole binding role was suggested for helix-8′. The activities and substrate K_ms of mutant α-subunits altered in this region by site-specific mutagenesis are reported here. Substitutions at Ser-235 by an acidic (glutamic acid, mutant SE235), basic (lysine, mutant SK235), or a nonpeptidyl amido-containing residue (proline, mutant SP235) exhibit 40- to 180-fold K_m increases for IGP and D -glyceraldehyde-3-phosphate; no K_m defects for indole were observed. k_cat values for SP235, SE235, and SK235 are 100, 70, and 40%, respectively, of the wild-type value. Steric considerations may explain the results with the SE235 and SK235 mutant α-subunits; however, the SP235 results are consistent with the suggested phosphate binding role for the Ser-235 peptidyl amide group during catalysis. A helix-8′ dipole role was explored following proline substitutions separately at the first six (of eight) residues. Proline substitutions at positions-1 through -4 in helix-8′ have normal indole K_ms and catalytic activities in all four TSase reactions, suggesting no major global structural changes in these proteins. By these criteria, substitutions at positions-5 and -6 lead to significant structural alterations. K_m increases for phosphorylated substrates are substantial (up to 40-fold) and are dependent upon the presence of L -serine at the β-subunit active site. In the absence of L -serine, substitution only at the first position results in binding defects; in the presence of L -serine, substitutions at the first, second and third positions show binding defects of decreasing magnitude, sequentially. Substitutions at the fourth and fifth position have no effect on substrate binding. It is suggested that during catalysis a helix dipole effect on binding may be exerted but only via inter-subunit-induced conformational changes due to ligand (L -serine) binding to the β-subunit. © 1995 Wiley-Liss, Inc.     

Role of Y(2) receptors in the regulation of gastric tone in rats

We set out to determine the effect of peptide YY(3-36) (PYY(3-36)) on the gastric muscle tone in conscious rats by measuring intragastric pressure (IGP) during intragastric nutrient drink infusion. After an overnight fast, a chronically implanted gastric fistula was connected to a custom-made nutrient drink infusion system and a catheter to measure IGP. IGP was measured before and during the infusion of a nutrient drink (Nutridrink; 0.5 ml/min) until 10 ml was infused. Rats were treated with PYY(3-36) (0, 33, and 100 pmol·kg(-1)·min(-1)) in combination with a subcutaneous injection of the Y(2) receptor antagonists JNJ31020028 (10 mg/kg) or BIIE0246 (2 mg/kg). Experiments were also performed after subdiaphragmatic vagotomy and after pretreatment with 3 ml of nutrient drink (to mimic a fed state). IGP was compared as the average IGP during nutrient infusion, represented as means ± SE and compared using ANOVA. PYY(3-36) dose dependently increased the IGP during nutrient infusion (4.7 ± 0.3, 5.7 ± 0.5 and 7.3 ± 0.7 mmHg; P < 0.01) while JNJ31020028 and BIIE0246 could block this increase [4.4 ± 0.5 (P < 0.001) and 4.8 ± 0.4 (P < 0.05) mmHg, respectively]. Also in vagotomized rats, PYY(3-36) was able to significantly increase the IGP during, an effect attenuated by JNJ31020028. BIIE0246 and JNJ31020028 were not able to decrease the IGP when no PYY(3-36) was administered. PYY(3-36) increased gastric tone through an Y(2) receptor-mediated mechanism that does not involve the vagus nerve. Y(2) receptor antagonists were not able to decrease gastric tone without exogenous administration of PYY(3-36), indicating that Y(2) receptors do not play a crucial role in the determination of gastric tone in physiological conditions.     

Synergistic effects on escape of a ligand from the closed tryptophan synthase bienzyme complex

Substrate channeling in the tryptophan synthase bienzyme complex is regulated by allosteric signals between the alpha- and beta-active sites acting over a distance of 25 A. At the alpha-site, indole is cleaved from 3-indole-D-glycerol 3'-phosphate (IGP) and is channeled to the beta-site via a tunnel. Harris and Dunn [Harris, R. M., and Dunn, M. F. (2002) Biochemistry 41, 9982-9990] showed that when the novel amino acid, dihydroiso-L-tryptophan (DIT), reacts with the beta-site, the alpha-aminoacrylate Schiff base, E(A-A), is formed and the enzyme releases indoline. The indoline produced exits the enzyme via the tunnel out the open alpha-site. When the alpha-site ligand (ASL) alpha-D,L-glycerol 3-phosphate (GP) binds and closes the alpha-site, indoline generated in the DIT reaction is trapped for a short period of time as the quinonoid intermediate in rapid equilibrium with bound indoline and the E(A-A) intermediate before leaking out of the closed enzyme. In this work, we use the DIT reaction and a new, high-affinity, ASL, N-(4-trifluoromethoxybenzenesulfonyl)-2-amino-1-ethyl phosphate (F9), to explore the mechanism of ligand leakage from the closed enzyme. It was found that F9 binding to the alpha-site is significantly more effective than GP in trapping indoline in the DIT reaction; however, leakage of indoline from the enzyme into solution still occurs. It was also found that a combination of benzimidazole (BZI) and GP provided even more effective trapping than F9. The new experiments with F9 and the combination of BZI and GP provide evidence that the coincident binding of GP and BZI at the alpha-site exhibits a strong synergistic effect that greatly slows the leakage of indoline in the DIT reaction and enhances the trapping effect. This synergism functions to tightly close the alpha-site and sends an allosteric signal that stabilizes the closed structure of the beta-site. These studies also support a mechanism for the escape of indoline through the alpha-site that is limited by ASL dissociation.     

Dynamics of a intraguild predation model with generalist or specialist predator

Intraguild predation (IGP) is a combination of competition and predation which is the most basic system in food webs that contains three species where two species that are involved in a predator/prey relationship are also competing for a shared resource or prey. We formulate two intraguild predation (IGP: resource, IG prey and IG predator) models: one has generalist predator while the other one has specialist predator. Both models have Holling-Type I functional response between resource-IG prey and resource-IG predator; Holling-Type III functional response between IG prey and IG predator. We provide sufficient conditions of the persistence and extinction of all possible scenarios for these two models, which give us a complete picture on their global dynamics. In addition, we show that both IGP models can have multiple interior equilibria under certain parameters range. These analytical results indicate that IGP model with generalist predator has “top down” regulation by comparing to IGP model with specialist predator. Our analysis and numerical simulations suggest that: (1) Both IGP models can have multiple attractors with complicated dynamical patterns; (2) Only IGP model with specialist predator can have both boundary attractor and interior attractor, i.e., whether the system has the extinction of one species or the coexistence of three species depending on initial conditions; (3) IGP model with generalist predator is prone to have coexistence of three species.     

蓝斑核和中缝背核参与电剌激弓状核引起的大鼠...

The effect of electrical stimulation of hypothalamic arcuate nucleus (ARC) on intragastric pressure (IGP) was observed on 80 Wistar rats anaesthetized with urethan. The main results are as follows: (1) Electrical stimulation of ARC could cause an obvious decrease of IGP. (2) The reduction of IGP induced by electrical stimulation of ARC was not affected by intracerebroventricular injection of naloxone. (3) After lesioning of locus coeruleus or dorsal raphe, the effect of ARC stimulation was depressed. The results suggest that the locus coeruleus and dorsal raphe nucleus may be involved in the reduction of IGP induced by ARC stimulation, but without the involvement of beta-endorphinergic neurons.