Regulation of tryptophan synthase by temperature, monovalent cations, and an allosteric ligand. Evidence from Arrhenius plots, absorption spectra, and primary kinetic isotope effects

To investigate the linkage between enzyme conformation and catalysis, we have determined the effects of temperature on catalytic properties of the tryptophan synthase alpha(2)beta(2) complex and beta(2) subunit in the absence or presence of different monovalent cations (Cs(+), Na(+), and GuH(+)) and of an allosteric ligand, alpha-glycerol 3-phosphate. Arrhenius plots of the activity data between 5 and 50 degrees C are nonlinear in the presence of certain ligands but not others. The conditions that yield nonlinear Arrhenius plots also yield temperature-dependent changes in the equilibrium distribution of enzyme-substrate intermediates and in primary kinetic isotope effects. The results provide evidence that the nonlinear Arrhenius plots are caused by a temperature-dependent conformational change that precedes the rate-limiting step in catalysis. Thermodynamic analysis of the data associated with the conformational change shows that the activation energies are much higher at low temperatures than at high temperatures. We correlate the results with a model in which the enzyme is converted by increased temperature under certain conditions from a low-activity "open" conformation to a high-activity "closed" conformation. The allosteric ligand and different monovalent cations, including GuH(+), which also acts as a chaotropic agent, affect the equilibrium between the open and closed forms. The large positive entropy changes in the conformational conversion suggest that the closed conformation results from tightened hydrophobic interactions that exclude water from the active site of the beta subunit.     

Allosteric regulation of tryptophan synthase channeling: the internal aldimine probed by trans-3-indole-3'-acrylate binding

Substrate channeling in the tryptophan synthase bienzyme complex from Salmonella typhimurium is regulated by allosteric interactions triggered by binding of ligand to the alpha-site and covalent reaction at the beta-site. These interactions switch the enzyme between low-activity forms with open conformations and high-activity forms with closed conformations. Previously, allosteric interactions have been demonstrated between the alpha-site and the external aldimine, alpha-aminoacrylate, and quinonoid forms of the beta-site. Here we employ the chromophoric l-Trp analogue, trans-3-indole-3'-acrylate (IA), and noncleavable alpha-site ligands (ASLs) to probe the allosteric properties of the internal aldimine, E(Ain). The ASLs studied are alpha-d,l-glycerol phosphate (GP) and d-glyceraldehyde 3-phosphate (G3P), and examples of two new classes of high-affinity alpha-site ligands, N-(4'-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) and N-(4'-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), that were previously shown to bind to the alpha-site by optical spectroscopy and X-ray crystal structures [Ngo, H., Harris, R., Kimmich, N., Casino, P., Niks, D., Blumenstein, L., Barends, T. R., Kulik, V., Weyand, M., Schlichting, I., and Dunn, M. F. (2007) Synthesis and characterization of allosteric probes of substrate channeling in the tryptophan synthase bienzyme complex, Biochemistry 46, 7713-7727]. The binding of IA to the beta-site is stimulated by the binding of GP, G3P, F6, or F9 to the alpha-site. The binding of ASLs was found to increase the affinity of the beta-site of E(Ain) for IA by 4-5-fold, demonstrating for the first time that the beta-subunit of the E(Ain) species undergoes a switching between low- and high-affinity states in response to the binding of ASLs.     

On the role of alphaThr183 in the allosteric regulation and catalytic mechanism of tryptophan synthase

The catalytic activity and substrate channeling of the pyridoxal 5'-phosphate-dependent tryptophan synthase alpha(2)beta(2) complex is regulated by allosteric interactions that modulate the switching of the enzyme between open, low activity and closed, high activity states during the catalytic cycle. The highly conserved alphaThr183 residue is part of loop alphaL6 and is located next to the alpha-active site and forms part of the alpha-beta subunit interface. The role of the interactions of alphaThr183 in alpha-site catalysis and allosteric regulation was investigated by analyzing the kinetics and crystal structures of the isosteric mutant alphaThr183Val. The mutant displays strongly impaired allosteric alpha-beta communication, and the catalytic activity of the alpha-reaction is reduced one hundred fold, whereas the beta-activity is not affected. The structural work establishes that the basis for the missing inter-subunit signaling is the lack of loop alphaL6 closure even in the presence of the alpha-subunit ligands, 3-indolyl-D-glycerol 3'-phosphate, or 3-indolylpropanol 3'-phosphate. The structural basis for the reduced alpha-activity has its origins in the missing hydrogen bond between alphaThr183 and the catalytic residue, alphaAsp60.     

Binding of monovalent cations induces large changes in the secondary structure of Na+,K+-ATPase as probed by Raman spectroscopy

Raman spectra of active Na+,K+-ATPase from pig kidney in media containing Na+ (E1), K+ (E2) or without exogenous ions (E1 conformation) were recorded in order to calculate the changes in the enzyme's secondary structure induced by binding of monovalent cations. It is demonstrated that: (i) K+ binding to the E1 form of the enzyme leads to conversion of approximately 100 peptide groups from the beta-structure to alpha-helical conformation; (ii) the transition is reversible and fully reproducible in the E1----E2----E1 and E2----E1----E2 experimental schemes. Predictional calculations revealed polypeptide chain segments involved in the alpha----beta transformations. These segments reside mainly in the two highly conserved regions of the alpha-subunit in the cytoplasmic domain of Na+,K+-ATPase. A possible role for the beta-subunit is discussed.     

Allosteric communication in the tryptophan synthase bienzyme complex: roles of the beta-subunit aspartate 305-arginine 141 salt bridge

The allosteric interactions that regulate substrate channeling and catalysis in the tryptophan synthase bienzyme complex from Salmonella typhimurium are triggered by covalent reactions at the beta-site and binding of substrate/product to the alpha-site. The transmission of these allosteric signals between the alpha- and beta-catalytic sites is modulated by an ensemble of weak bonding interactions consisting of salt bridges, hydrogen bonds, and van der Waals contacts that switch the subunits between open and closed conformations. Previous work has identified a scaffolding of salt-bridges extending between the alpha- and beta-sites consisting of alphaAsp 56, betaLys 167, and betaAsp 305. This work investigates the involvement of yet another salt bridging interaction involving the betaAsp 305-betaArg 141 pair via comparison of the spectroscopic, catalytic, and allosteric properties of the betaD305A and betaR141A mutants with the behavior of the wild-type enzyme. These mutations were found to give bienzyme complexes with impaired allosteric communication. The betaD305A mutant also exhibits altered beta-site substrate reaction specificity, while the catalytic activity of the betaR141A mutant exhibits impaired beta-site catalytic activity. The >25-fold activation of the alpha-site by alpha-aminoacrylate Schiff base formation at the beta-site found in the Na(+) form of the wild-type enzyme is abolished in the Na(+) forms of both mutants. Replacing Na(+) by NH(4)(+) or Cs(+) restores the betaD305A to a wild-type-like behavior, whereas only partial restoration is achieved with the betaR141A mutant. These studies establish that the betaD305-betaR141 salt bridge plays a crucial role both in the formation of the closed conformation of the beta-site and in the transmission of allosteric signals between the alpha- and beta-sites that switch the alpha-site on and off.     

The ATPase activity of saponin-treated rat erythrocytes: regulation by monovalent cations, calcium, ouabain, and furosemide

The ATPase activities were studied in rat erythrocytes permeabilized with saponin. The concentrations of calcium and magnesium ions were varied within the range of 0.1-60 microM and 50-370 microM, respectively, by using EGTA-citrate buffer. The maximal activity of Ca2(+)-ATPase of permeabilized erythrocytes was by one order of magnitude higher, whereas the Ca2(+)-binding affinity was 1.5-2 times higher than that in erythrocyte ghosts washed an isotonic solution containing EGTA. Addition of the hemolysate restored the kinetic parameters of ghost Ca2(+)-ATPase practically completely, whereas in the presence of exogenous calmodulin only part of Ca2(+)-ATPase activity was recovered. Neither calmodulin nor R24571, a highly potent specific inhibitor of calmodulin-dependent reactions, influenced the Ca2(+)-ATPase activity of permeabilized erythrocytes. At Ca2+ concentrations below 0.7 microM, ouabain (0.5-1 mM) activated whereas at higher Ca2+ concentrations it inhibited the Ca2(+)-ATPase activity. Taking this observation into account the Na+/K(+)-ATPase was determined as the difference of between the ATPase activities in the presence of Na+ and K+ and in the presence of K+ alone. At physiological concentration of Mg2+ (370 microM), the addition of 0.3-1 microM Ca2+ increased Na+/K(+)-ATPase activity by 1.5-3-fold. Higher concentrations of this cation inhibited the enzyme. At low Mg2+ concentration (e.g., 50 microM) only Na+/K(+)-ATPase inhibition by Ca2+ was seen. It was found that at [NaCl] less than 20 mM furosemide was increased ouabain-inhibited component of ATPase in Ca2(+)-free media. This activating effect of furosemide was enhanced with a diminution of [Na+] upto 2 mM and did not reach the saturation level unless the 2 mM of drug was used. The activating effect of furosemide on Na+/K(+)-ATPase activity confirmed by experiments in which the ouabain-inhibited component was measured by the 86Rb+ influx into intact erythrocytes.     

Mechanisms of monovalent cation action in enzyme catalysis: the tryptophan synthase alpha-, beta-, and alpha beta-reactions.

The alpha-subunit of the tryptophan synthase bienzyme complex catalyzes the formation of indole from the cleavage of 3-indolyl-D-glyceraldehyde 3'-phosphate, while the beta-subunit utilizes L-serine and the indole produced at the alpha-site to form tryptophan. The replacement reaction catalyzed by the beta-subunit requires pyridoxal 5'-phosphate (PLP) as a cofactor. The beta-reaction occurs in two stages: in stage I, the first substrate, L-Ser, reacts with the enzyme-bound PLP cofactor to form an equilibrating mixture of the L-Ser Schiff base, E(Aex1), and the alpha-aminoacrylate Schiff base intermediate, E(A-A); in stage II, this intermediate reacts with the second substrate, indole, to form tryptophan. Monovalent cations (MVCs) are effectors of these processes [Woehl, E., and Dunn, M. F. (1995) Biochemistry 34, 9466-9476]. Herein, detailed kinetic dissections of stage II are described in the absence and in the presence of MVCs. The analyses presented complement the results of the preceding paper [Woehl, E., and Dunn, M. F. (1999) Biochemistry 38, XXXX-XXXX], which examines stage I, and confirm that the chemical and conformational processes in stage I establish the presence of two slowly interconverting conformations of E(A-A) that exhibit different reactivities in stage II. The pattern of kinetic isotope effects on the overall activity of the beta-reaction shows an MVC-mediated change in rate-limiting steps. In the absence of MVCs, the reaction of E(A-A) with indole becomes the rate-limiting step. In the presence of Na+ or K+, the conversion of E(Aex1) to E(A-A) is rate limiting, whereas some third process not subject to an isotope effect becomes rate determining for the NH4+-activated enzyme. The combined results from the preceding paper and from this study define the MVC effects, both for the reaction catalyzed by the beta-subunit and for the allosteric communication between the alpha- and beta-sites. Partial reaction-coordinate free energy diagrams and simulation studies of MVC effects on the proposed mechanism of the beta-reaction are presented.     

Effects of temperature, pH, and salt concentration on beta-lactoglobulin deposition kinetics studied by optical waveguide lightmode spectroscopy

Deposition kinetics of beta-lactoglobulin at a solid-liquid interface was studied with optical waveguide lightmode spectroscopy (OWLS) over a range of temperatures between 61 and 83 degrees C. A new temperature-controlled cell for OWLS measurements allows fast, on-line monitoring of the deposit formation at elevated temperatures. Primary protein layers were deposited at 25 degrees C in order to precondition and stabilize the waveguide surface. Sustained deposition lasting from a few minutes (around 80 degrees C) to hours (below 70 degrees C) resulted in multilayer deposits up to several tens of nanometers thick. The measured deposition rates were strongly influenced by temperature, pH, and NaCl concentration. Deposition rates decreased with increasing pH from 5.5. to 7.4, in a trend similar to that for noncovalent aggregation of beta-lactoglobulin in solution. Activation energies for deposition rates decreased with increasing pH, from 340 kJ/mol at pH 5.5 to 230 kJ/mol at pH 7.4 and were similar to the activation energies for denaturation of beta-lactoglobulin in solution.     

Structural and dynamic properties of the heme pocket in myoglobin probed by optical spectroscopy

We report the optical absorption spectra of sperm whale deoxy-, oxy-, and carbonmonoxymyoglobin in the temperature range 300–20 K and in 65% glycerol or ethylene glycol–water mixtures. By lowering the temperature, all bands exhibit half-width narrowing and peak frequency shift; moreover, the near-ir bands of deoxymyoglobin show a marked increase of the integrated intensities. Opposed to what has already been reported for human hemoglobin, the temperature dependence of the first moment of the investigated bands does not follow the behavior predicted by the harmonic Franck–Condon approximation and is sizably affected by the solvent composition; this solvent effect is larger in liganded than in nonliganded myoglobin. However, for all the observed bands the behavior of the second moment can be quite well rationalized in terms of the harmonic Franck–Condon approximation and is not dependent on solvent composition. On the basis of these data we put forward some suggestions concerning the structural and dynamic properties of the heme pocket in myoglobin and their dependence upon solvent composition. We also discuss the different behaviors of myoglobin and hemoglobin in terms of the different heme pocket structures and deformabilities of the two proteins.     

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.     

Mechanisms of monovalent cation action in enzyme catalysis: the first stage of the tryptophan synthase beta-reaction.

The tryptophan synthase bienzyme complex is activated and regulated by the allosteric action of monovalent cations (MVCs). The kinetic dissection of the first stage (stage I) in the beta-reaction of tryptophan synthase, the reaction of L-serine with pyridoxal 5'-phosphate at the beta-site to give the alpha-aminoacrylate Schiff base intermediate, E(A-A), is here examined in the absence and presence of MVCs. This analysis reveals which of the individual steps are greatly affected in stage I and how the ground states and transition states are affected by MVCs. Kinetic studies in combination with a detailed relaxation kinetic analysis to determine the specific rate constants for the conversion of the L-Ser external aldimine, E(Aex1), to E(A-A) show that the primary kinetic isotope effect for proton abstraction from Calpha of the E(Aex1) species changes from 4.0 +/- 0.4 in the absence of MVCs to a value of 5.9 +/- 0.5 in the presence of Na+, indicating that the nature of the transition state for this C-H scission is significantly perturbed by the MVC effect. The E(A-A) species was found to exist in two conformations with different activities, the ratio of which is affected by the presence of MVCs. It is shown that changes in the rate constants of stage I are important in establishing the ratio of active to inactive conformations of the E(A-A) species. Consequently, the MVC effect alters the relative energies of both the transition states and the ground states for selected steps in stage I of the pathway. Hence, interactions at the MVC site give rise to a fine-tuning of the covalent bonding interactions between active site residues and the reacting substrate during the conformational cycle of the bienzyme complex.     

HlyC, the internal protein acyltransferase that activates hemolysin toxin: roles of various conserved residues in enzymatic activity as probed by site-directed mutagenesis.

Hemolysin, a toxic protein produced by pathogenic Escherichia coli, is one of a family of homologous toxins and toxin-processing proteins produced by Gram-negative bacteria. HlyC, an internal protein acyltransferase, converts it from nontoxic prohemolysin to toxic hemolysin. Acyl-acyl carrier protein is the essential acyl donor. The acyltransferase reaction progresses through formation of a binary complex between acyl-ACP and HlyC to a reactive acyl-HlyC intermediate [Trent, M. S., Worsham, L. M., and Ernst-Fonberg, M. L. (1998) Biochemistry 37, 4644-4655]. The homologous acyltransferases of the family have a number of conserved amino acid residues that may be catalytically important. Experiments to illuminate the reaction mechanism were done. The formation of an acyl-enzyme intermediate suggested that the reaction likely proceeded through two partial reactions. The reversibility of the first partial reaction was shown by using separately subcloned, purified, and expressed substrates and enzyme. The effects of single site-directed mutations of conserved residues of HlyC on different portions of reaction progress (binary complex formation, acyl-enzyme formation, and enzyme activity, including kinetic parameters) were determined. Mutations of His23, the only residue essential for activity, formed normal binary complexes but were unable to form acyl-HlyC. The same was seen with S20A, a mutant with greatly impaired activity. Mutation of two conserved tyrosines separately to glycines results in greatly impaired binary complex and acyl-HlyC formation, but mutation of those residues to phenylalanines restored behavior to wild-type.     

The binding mechanism,multiple binding modes,and allosteric regulation of Staphylococcus aureus Sortase A probed by molecular dynamics simulations

Sortase enzymes are vitally important for the virulence of gram‐positive bacteria as they play a key role in the attachment of surface proteins to the cell wall. These enzymes recognize a specific sorting sequence in proteins destined to be displayed on the surface of the bacteria and catalyze the transpeptidation reaction that links it to a cell wall precursor molecule. Because of their role in establishing pathogenicity, and in light of the recent rise of antibiotic‐resistant bacterial strains, sortase enzymes are novel drug targets. Here, we present a study of the prototypical sortase protein Staphylococcus aureus Sortase A (SrtA). Both conventional and accelerated molecular dynamics simulations of S. aureus SrtA in its apo state and when bound to an LPATG sorting signal (SS) were performed. Results support a binding mechanism that may be characterized as conformational selection followed by induced fit. Additionally, the SS was found to adopt multiple metastable states, thus resolving discrepancies between binding conformations in previously reported experimental structures. Finally, correlation analysis reveals that the SS actively affects allosteric pathways throughout the protein that connect the first and the second substrate binding sites, which are proposed to be located on opposing faces of the protein. Overall, these calculations shed new light on the role of dynamics in the binding mechanism and function of sortase enzymes.     

The allosteric transition in DnaK probed by infrared difference spectroscopy. Concerted ATP-induced rearrangement of the substrate binding domain

The biological activity of DnaK, the bacterial representative of the Hsp70 protein family, is regulated by the allosteric interaction between its nucleotide and peptide substrate binding domains. Despite the importance of the nucleotide-induced cycling of DnaK between substrate-accepting and releasing states, the heterotropic allosteric mechanism remains as yet undefined. To further characterize this mechanism, the nucleotide-induced absorbance changes in the vibrational spectrum of wild-type DnaK was characterized. To assign the conformation sensitive absorption bands, two deletion mutants (one lacking the C-terminal alpha-helical subdomain and another comprising only the N-terminal ATPase domain), and a single-point DnaK mutant (T199A) with strongly reduced ATPase activity, were investigated by time-resolved infrared difference spectroscopy combined with the use of caged-nucleotides. The results indicate that (1) ATP, but not ADP, binding promotes a conformational change in both subdomains of the peptide binding domain that can be individually resolved; (2) these conformational changes are kinetically coupled, most likely to ensure a decrease in the affinity of DnaK for peptide substrates and a concomitant displacement of the lid away from the peptide binding site that would promote efficient diffusion of the released peptide to the medium; and (3) the alpha-helical subdomain contributes to stabilize the interdomain interface against the thermal challenge and allows bidirectional transmission of the allosteric signal between the ATPase and substrate binding domains at stress temperatures (42 degrees C).     

Effect of pH and monovalent cations on the formation of quinonoid intermediates of the tryptophan synthase alpha(2)beta(2) complex in solution and in the crystal

Quinonoid intermediates play a key role in the catalytic mechanism of pyridoxal 5'-phosphate-dependent enzymes. Whereas the structures of other pyridoxal 5'-phosphate-bound intermediates have been determined, the structure of a quinonoid species has not yet been reported. Here, we investigate factors controlling the accumulation and stability of quinonoids formed at the beta-active site of tryptophan synthase both in solution and the crystal. The quinonoids were obtained by reacting the alpha-aminoacrylate Schiff base with different nucleophiles, focusing mainly on the substrate analogs indoline and beta-mercaptoethanol. In solution, both monovalent cations (Cs(+) or Na(+)) and alkaline pH increase the apparent affinity of indoline and favor accumulation of the indoline quinonoid. A similar pH dependence is observed when beta-mercaptoethanol is used. As indoline and beta-mercaptoethanol exhibit very distinct ionization properties, this finding suggests that nucleophile binding and quinonoid stability are controlled by some ionizable protein residue(s). In the crystal, alkaline pH favors formation of the indoline quinonoid as in solution, but the effect of cations is markedly different. In the absence of monovalent metal ions the quinonoid species accumulates substantially, whereas in the presence of sodium ions the accumulation is modest, unless alpha-subunit ligands are also present. Alpha-subunit ligands not only favor the formation of the intermediate, but also reduce significantly its decay rate. These findings define experimental conditions suitable for the stabilization of the quinonoid species in the crystal, a critical prerequisite for the determination of the three-dimensional structure of this intermediate.     

Pyruvate kinase: Activation by and catalytic role of the monovalent and divalent cations

Summary This mini review is primarily concerned with the monovalent and divalent cation activation of pyruvate kinase. All preparations of pyruvate kinase from vertebrate tissue which have been examined require monovalent cations such as K⁺ for catalysis. However, several microbial preparations are not activated by monovalent cations. In fact,E. coli synthesizes depending on growth conditions, 2 different forms of the enzyme; one form is not activated while the other is activated by monovalent cations. The monovalent cation was shown by NMR techniques to bind within 4–8 ? of the divalent cation activat or and apparently plays a direct role in the catalytic process. As with all kinases, pyruvate kinase requires a divalent cation for catalysis. Mg⁺² is optimal for the physiological reaction, however, Co⁺², Mn⁺², and Ni⁺² also activate. The divalent cation activation of several non-physiological reactions catalyzed by pyruvate kinase are reviewed. Several lines of evidence suggest that 2 moles of the divalent cation are required in the catalytic event. However, the specific role of both atoms in the catalytic event have not been thoroughly elucidated.     

Two anthranilate synthase genes in Arabidopsis: defense-related regulation of the tryptophan pathway.

Arabidopsis thaliana has two genes, ASA1 and ASA2, encoding the alpha subunit of anthranilate synthase, the enzyme catalyzing the first reaction in the tryptophan biosynthetic pathway. As a branchpoint enzyme in aromatic amino acid biosynthesis, anthranilate synthase has an important regulatory role. The sequences of the plant genes are homologous to their microbial counterparts. Both predicted proteins have putative chloroplast transit peptides at their amino termini and conserved amino acids involved in feedback inhibition by tryptophan. ASA1 and ASA2 cDNAs complement anthranilate synthase alpha subunit mutations in the yeast Saccharomyces cerevisiae and in Escherichia coli, confirming that both genes encode functional anthranilate synthase proteins. The distributions of ASA1 and ASA2 mRNAs in various parts of Arabidopsis plants are overlapping but nonidentical, and ASA1 mRNA is approximately 10 times more abundant in whole plants. Whereas ASA2 is expressed at a constitutive basal level, ASA1 is induced by wounding and bacterial pathogen infiltration, suggesting a novel role for ASA1 in the production of tryptophan pathway metabolites as part of an Arabidopsis defense response. Regulation of key steps in aromatic amino acid biosynthesis in Arabidopsis appears to involve differential expression of duplicated genes.     

A conserved tryptophan 457 modulates the kinetics and extent of N-hydroxy-L-arginine oxidation by inducible nitric-oxide synthase

In the oxygenase domain of mouse inducible nitric-oxide synthase (iNOSoxy), a conserved tryptophan residue, Trp-457, regulates the kinetics and extent of l-Arg oxidation to N(omega)-hydroxy-l-arginine (NOHA) by controlling electron transfer between bound (6R)-tetrahydrobiopterin (H(4)B) cofactor and the enzyme heme Fe(II)O(2) intermediate (Wang, Z. Q., Wei, C. C., Ghosh, S., Meade, A. L., Hemann, C., Hille, R., and Stuehr, D. J. (2001) Biochemistry 40, 12819-12825). To investigate whether NOHA oxidation to citrulline and nitric oxide (NO) is regulated by a similar mechanism, we performed single turnover reactions with wild type iNOSoxy and mutants W457F and W457A. Ferrous proteins containing NOHA plus H(4)B or NOHA plus 7,8-dihydrobiopterin (H(2)B), were mixed with O(2)-containing buffer, and then heme spectral transitions and product formation were followed versus time. All three proteins formed a Fe(II)O(2) intermediate with identical spectral characteristics. In wild type, H(4)B increased the disappearance rate of the Fe(II)O(2) intermediate relative to H(2)B, and its disappearance was coupled to the formation of a Fe(III)NO immediate product prior to formation of ferric enzyme. In W457F and W457A, the disappearance rate of the Fe(II)O(2) intermediate was slower than in wild type and took place without detectable build-up of the heme Fe(III)NO immediate product. Rates of Fe(II)O(2) disappearance correlated with rates of citrulline formation in all three proteins, and reactions containing H(4)B formed 1.0, 0.54, and 0.38 citrulline/heme in wild type, W457F, and W457A iNOSoxy, respectively. Thus, Trp-457 modulates the kinetics of NOHA oxidation by iNOSoxy, and this is important for determining the extent of citrulline and NO formation. Our findings support a redox role for H(4)B during NOHA oxidation to NO by iNOSoxy.     

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.