Monovalent Cations Partially Repair a Conformational Defect in a Mutant Tryptophan Synthase α2β2 Complex (β-E109A)
1995; Elsevier BV; Volume: 270; Issue: 29 Linguagem: Inglês
10.1074/jbc.270.29.17333
ISSN1083-351X
AutoresSergei B. Ruvinov, S. Ashraf Ahmed, Peter McPhie, Edith Wilson Miles,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoWe are using the tryptophan synthase α2β2 complex as a model system to investigate how ligands, protein-protein interaction, and mutations regulate enzyme activity, reaction specificity, and substrate specificity. The rate of conversion of L-serine and indole to L-tryptophan by the β2 subunit alone is quite low, but is activated by certain monovalent cations or by association with α subunit to form an α2β2 complex. Since monovalent cations and α subunit appear to stabilize an active conformation of the β2 subunit, we have investigated the effects of monovalent cations on the activities and spectroscopic properties of a mutant form of α2β2 complex having β2 subunit glutamic acid 109 replaced by alanine (E109A). The E109A α2β2 complex is inactive in reactions with L-serine but active in reactions with β-chloro-L-alanine. Parallel experiments show effects of monovalent cations on the properties of wild type β2 subunit and α2β2 complex. We find that CsCl stimulates the activity of the E109A α2β2 complex and of wild type β2 subunit with L-serine and indole and alters the equilibrium distribution of L-serine reaction intermediates. The results indicate that CsCl partially repairs the deleterious effects of the E109A mutation on the activity of the α2β2 complex by stabilizing a conformation with catalytic properties more similar to those of the wild type α2β2 complex. This conclusion is consistent with observations that monovalent cations alter the catalytic and spectroscopic properties of several pyridoxal phosphate-dependent enzymes by stabilizing alternative conformations. We are using the tryptophan synthase α2β2 complex as a model system to investigate how ligands, protein-protein interaction, and mutations regulate enzyme activity, reaction specificity, and substrate specificity. The rate of conversion of L-serine and indole to L-tryptophan by the β2 subunit alone is quite low, but is activated by certain monovalent cations or by association with α subunit to form an α2β2 complex. Since monovalent cations and α subunit appear to stabilize an active conformation of the β2 subunit, we have investigated the effects of monovalent cations on the activities and spectroscopic properties of a mutant form of α2β2 complex having β2 subunit glutamic acid 109 replaced by alanine (E109A). The E109A α2β2 complex is inactive in reactions with L-serine but active in reactions with β-chloro-L-alanine. Parallel experiments show effects of monovalent cations on the properties of wild type β2 subunit and α2β2 complex. We find that CsCl stimulates the activity of the E109A α2β2 complex and of wild type β2 subunit with L-serine and indole and alters the equilibrium distribution of L-serine reaction intermediates. The results indicate that CsCl partially repairs the deleterious effects of the E109A mutation on the activity of the α2β2 complex by stabilizing a conformation with catalytic properties more similar to those of the wild type α2β2 complex. This conclusion is consistent with observations that monovalent cations alter the catalytic and spectroscopic properties of several pyridoxal phosphate-dependent enzymes by stabilizing alternative conformations. The regulation of enzyme activity and of substrate specificity is a problem of critical importance in biology. Enzyme activity can be modulated by interaction of the enzyme with ligands or with other proteins. These interactions produce changes in protein conformation or alter the equilibrium distribution of preexisting conformations. Mutations can also modulate enzyme activity by altering protein conformation. In the present work we ask whether ligands that stabilize the active conformation of an enzyme can also repair the deleterious effects of a mutation that leads to an inactive conformation. Tryptophan synthase (EC 4.2.1.20) is a classic system for investigating how ligands, protein-protein interaction, and mutations regulate enzyme activity and substrate specificity (for reviews, see (1Yanofsky C. Crawford I.P. Boyer P.D. The Enzymes. 3rd Ed. Academic Press, New York1972: 1-31Google Scholar, 2Miles E.W. Adv. Enzymol. 1979; 49: 127-186PubMed Google Scholar, 3Miles E.W. Adv. Enzymol. Relat. Areas Mol. Biol. 1991; 64: 93-172PubMed Google Scholar, 4Miles E.W. Ahmed S.A. Hyde C.C. Kayastha A.M. Yang X.-J. Ruvinov S.B. Lu Z. Fukui T. Soda K. Molecular Aspects of Enzyme Catalysis. Kodansha, Ltd., Tokyo, Japan1994: 127-146Crossref Scopus (3) Google Scholar, 5Miles E.W. Biswas B.B. Roy S. Subcellular Biochemistry; Proteins: Structure, Function, and Protein Engineering. Plenum Press, New York1995: 207-254Google Scholar)). The bacterial enzyme is an α2β2 complex that dissociates reversibly into monomeric α subunits and dimeric β2 subunits. The separate α subunit catalyzes the reversible aldolytic cleavage of indole-3-glycerol phosphate to D-glyceraldehyde 3-phosphate and indole, termed the α reaction. The separate β2 subunit catalyzes the condensation of L-serine with indole to form L-tryptophan in a pyridoxal phosphate-dependent β-replacement reaction, termed the β reaction. The interaction of the α and β2 subunits in the α2β2 complex serves to increase the rates of the α and β reactions up to 100-fold, increase substrate binding affinities, and alter reaction specificities. These changes are attributed to conformational changes which occur upon assembly of the complex(6Lane A.N. Paul C.H. Kirschner K. EMBO J. 1984; 3: 279-287Crossref PubMed Scopus (32) Google Scholar). The binding of a ligand or the formation of a reaction intermediate at the active site of either subunit in the α2β2 complex alters the reaction kinetics at the heterologous active site ∼25 Å distant(7Lane A.N. Kirschner K. Eur. J. Biochem. 1983; 129: 561-570Crossref PubMed Scopus (72) Google Scholar, 8Houben K.F. Dunn M.F. Biochemistry. 1990; 29: 2421-2429Crossref PubMed Scopus (63) Google Scholar, 9Drewe Jr., W.F. Dunn M.F. Biochemistry. 1985; 24: 3977-3987Crossref PubMed Scopus (95) Google Scholar, 10Creighton T.E. Eur. J. Biochem. 1970; 13: 1-10Crossref PubMed Scopus (84) Google Scholar, 11Brzovic' P.S. Ngo K. Dunn M.F. Biochemistry. 1992; 31: 3831-3839Crossref PubMed Scopus (96) Google Scholar, 12Dunn M.F. Aguilar V. Brzovic' P.S. Drewe Jr., W.F. Houben K.F. Leja C.A. Roy M. Biochemistry. 1990; 29: 8598-8607Crossref PubMed Scopus (151) Google Scholar, 13Dunn M.F. Aguilar V. Drewe W.J. Houben K. Robustell B. Roy M. Indian J. Biochem. Biophys. 1987; 24: 44-51Google Scholar, 14Kawasaki H. Bauerle R. Zon G. Ahmed S.A. Miles E.W. J. Biol. Chem. 1987; 262: 10678-10683Abstract Full Text PDF PubMed Google Scholar, 15Kirschner K. Lane A.N. Strasser A.W.M. Biochemistry. 1991; 30: 472-478Crossref PubMed Scopus (72) Google Scholar, 16Anderson K.S. Miles E.W. Johnson K.A. J. Biol. Chem. 1991; 266: 8020-8033Abstract Full Text PDF PubMed Google Scholar). The combined results suggest that the α and β2 subunits undergo conformational changes during catalysis and exist in two or more conformations that have been designated "open" and "closed" (11Brzovic' P.S. Ngo K. Dunn M.F. Biochemistry. 1992; 31: 3831-3839Crossref PubMed Scopus (96) Google Scholar). Investigations of the reaction and substrate specificities of wild type and mutant forms of the β2 subunit and the α2β2 complex also provide evidence that the β2 subunit can exist in two different conformational states (designated I and II in Table 1)(17Ahmed S.A. Ruvinov S.B. Kayastha A.M. Miles E.W. J. Biol. Chem. 1991; 266: 21548-21557Abstract Full Text PDF PubMed Google Scholar, 18Brzovic' P.S. Kayastha A.M. Miles E.W. Dunn M.F. Biochemistry. 1992; 31: 1180-1190Crossref PubMed Scopus (51) Google Scholar). The separate β2 subunit catalyzes β-replacement and β-elimination reactions with a variety of amino acids that have an electronegative substituent in the β-position (e.g.L-serine and β-chloro-L-alanine)(19Kumagai H. Miles E.W. Biochem. Biophys. Res. Commun. 1971; 44: 1271-1278Crossref PubMed Scopus (47) Google Scholar, 20Miles E.W. Dolphin D. Poulson D. Avramovic O. Pyridoxal Phosphate and Derivatives. John Wiley and Sons, New York1986: 253-310Google Scholar).TABLE I Open table in a new tab β-replacement reaction: RCH2CHNH2COOH+R'H↔R'CH2CHNH2COOH+RH(Eq. 1) β-replacement reaction: RCH2CHNH2COOH+H2O→CH3COCOOH+RH+NH3(Eq. 2) Association of the β2 subunit with the α subunit greatly increases activity in the β-replacement reaction with L-serine and indole and almost completely eliminates activity in the β-elimination reaction with L-serine. Thus association with the α subunit alters the reaction specificity of the β2 subunit. Whereas L-serine is a better substrate than β-chloro-L-alanine for the β-replacement reaction with indole catalyzed by the α2β2 complex, the reverse is true for the β2 subunit(17Ahmed S.A. Ruvinov S.B. Kayastha A.M. Miles E.W. J. Biol. Chem. 1991; 266: 21548-21557Abstract Full Text PDF PubMed Google Scholar). Thus association with the α subunit alters the substrate specificity of the β2 subunit. Several α2β2 complexes with single amino acid replacements in the active site of the β2 subunit have substrate and reaction specificities similar to those of the wild type β2 subunit(17Ahmed S.A. Ruvinov S.B. Kayastha A.M. Miles E.W. J. Biol. Chem. 1991; 266: 21548-21557Abstract Full Text PDF PubMed Google Scholar). This finding led us to suggest that the wild type β2 subunit and these mutant α2β2 complexes exist in open conformations (I in Table 1), whereas the wild type α2β2 complex exists in a closed conformation (II in Table 1)(17Ahmed S.A. Ruvinov S.B. Kayastha A.M. Miles E.W. J. Biol. Chem. 1991; 266: 21548-21557Abstract Full Text PDF PubMed Google Scholar). Aliphatic alcohols also stabilize an alternative conformation of the α2β2 complex that has properties similar to that of the free β2 subunit in aqueous solution (21Ahmed S.A. Miles E.W. J. Biol. Chem. 1994; 269: 16486-16492Abstract Full Text PDF PubMed Google Scholar) (I in Table 1). Early studies showed that some monovalent cations (K+, Li+, and NH4+, but not Na+) activate the β2 subunit (22Crawford I.P. Ito J. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 390-397Crossref PubMed Scopus (47) Google Scholar, 23Hatanaka M. White E.A. Horibata K. Crawford I.P. Arch. Biochem. Biophys. 1962; 97: 596-606Crossref PubMed Scopus (39) Google Scholar) in β-replacement and β-elimination reactions with L-serine. High concentrations of NH4+ in some ways mimic the effects of the α subunit on the kinetic and spectroscopic properties of the β2 subunit(24Goldberg M.E. York S. Stryer L. Biochemistry. 1968; 7: 3662-3667Crossref PubMed Scopus (76) Google Scholar, 25Miles E.W. McPhie P. Biochemistry. 1974; 249: 2852-2857Google Scholar, 26York S.S. Biochemistry. 1972; 11: 2733-2740Crossref PubMed Scopus (42) Google Scholar). Recently it has been shown that monovalent cations (Na+, K+, Li+, Rb+, NH4+, and Cs+) activate the wild type tryptophan synthase α2β2 complex and alter the pH-dependent equilibrium distribution of enzyme-substrate intermediates(27Dunn M.F. Brzovic' P.S. Leja C. Pan P. Woehl E.U. Marino G. Sannia G. Bossa F. Biochemistry of Vitamin B and PQQ. Birkhauser Verlag, Basel/Switzerland1994: 119-124Google Scholar, 28Peracchi A. Mozzarelli A. Rossi G.L. Marino G. Sannia G. Bossa F. Biochemistry of Vitamin B and PQQ. Birkhauser Verlag, Basel/Switzerland1994: 125-129Google Scholar, 29Peracchi A. Mozzarelli A. Rossi G.L. Biochemistry. 1995; (in press)PubMed Google Scholar, 30Woehl E.U. Dunn M.F. Coordination Chemistry Reviews. 1995; (in press)Google Scholar, 31Woehl E.U. Dunn M.F. Biochemistry. 1995; (in press)Google Scholar). Together the results support the idea that both the free β2 subunit and the α2β2 complex exist in two or more conformations. Preferential binding of certain monovalent cations to a specific site in the β2 subunit may alter the equilibrium distribution of preexisting conformations in the β2 subunit and α2β2 complex by stabilizing conformation II (Table 1). Binding of an α subunit ligand (α-glycerol 3-phosphate) to the α2β2 complex also alters the distribution of intermediates formed with L-serine(32Mozzarelli A. Peracchi A. Bettati S. Rossi G.L. Fukui T. Kagamiyama H. Soda K. Wada H. Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds as Cofactors. Pergamon Press, Oxford1991: 273-275Crossref Google Scholar). Table 1 indicates that α site ligands stabilize conformation II. Because certain cations and α site ligands appear to stabilize the more active conformation II of wild type β2 subunit and α2β2 complex (Table 1), we reasoned that these ligands might promote the conversion of a mutant α2β2 complex from an inactive conformation I to an active conformation II. In the present work we have investigated the effects of monovalent cations on a mutant α2β2 complex having Glu-109 of the β2 subunit replaced by alanine (E109A). Glu-109 is located in a region of the β2 subunit adjacent to the covalently bound pyridoxal phosphate cofactor and near the putative indole-binding site of the β2 subunit (see Fig. 1 in (17Ahmed S.A. Ruvinov S.B. Kayastha A.M. Miles E.W. J. Biol. Chem. 1991; 266: 21548-21557Abstract Full Text PDF PubMed Google Scholar)). It has been postulated that the carboxylate of Glu-109 may activate the indole toward nucleophilic attack on the aminoacrylate (33Kayastha A.M. Miles E.W. FASEB J. 1990; 4: A2118Google Scholar) and/or activation of the β-hydroxyl of L-serine as a leaving group(18Brzovic' P.S. Kayastha A.M. Miles E.W. Dunn M.F. Biochemistry. 1992; 31: 1180-1190Crossref PubMed Scopus (51) Google Scholar). The finding that the E109A α2β2 complex can synthesize L-tryptophan from β-chloro-L-alanine and indole but not from L-serine and indole provides evidence that Glu-109 is not an essential residue for the activation of indole but may serve to activate the β hydroxyl of L-serine as a leaving group(17Ahmed S.A. Ruvinov S.B. Kayastha A.M. Miles E.W. J. Biol. Chem. 1991; 266: 21548-21557Abstract Full Text PDF PubMed Google Scholar, 18Brzovic' P.S. Kayastha A.M. Miles E.W. Dunn M.F. Biochemistry. 1992; 31: 1180-1190Crossref PubMed Scopus (51) Google Scholar, 34Kayastha A.M. Miles E.W. Fukui T. Kagamiyama H. Soda K. Wada H. Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds as Cofactors. Pergamon Press, Oxford1991: 265-267Crossref Google Scholar). Alternatively, replacement of Glu-109 by alanine (17Ahmed S.A. Ruvinov S.B. Kayastha A.M. Miles E.W. J. Biol. Chem. 1991; 266: 21548-21557Abstract Full Text PDF PubMed Google Scholar) or aspartate (16Anderson K.S. Miles E.W. Johnson K.A. J. Biol. Chem. 1991; 266: 8020-8033Abstract Full Text PDF PubMed Google Scholar, 18Brzovic' P.S. Kayastha A.M. Miles E.W. Dunn M.F. Biochemistry. 1992; 31: 1180-1190Crossref PubMed Scopus (51) Google Scholar) may introduce structural changes within the β2 subunit that alter the conformation of the α2β2 complex and thereby alter the substrate and reaction specificity. Our finding that the E109A α2β2 complex resembles the wild type β2 subunit in substrate specificity and reaction specificity suggests that the mutation stabilizes conformation I (see "mutations" in Table 1). The results reported here show that addition of a high concentration of CsCl partially restores the activity of the E109A α2β2 complex with L-serine and alters the substrate and reaction specificity. The results suggest that CsCl repairs a conformational defect in the E109A α2β2 complex by stabilizing conformation II that has structural and catalytic properties more similar to those of the wild type α2β2 complex. DL-α-Glycerol 3-phosphate (GP),1 1The abbreviations used are: GPDL-α-glycerol 3-phosphatebis-tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol. β-chloro-L-alanine (hydrochloride), and pyridoxal phosphate were from Sigma. All spectroscopic experiments and enzyme assays utilized Buffer B (50 mM sodium N,N-bis(2-hydroxyethyl)glycine containing 1 mM EDTA at pH 7.8). DL-α-glycerol 3-phosphate 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol. Wild type (35Miles E.W. Kawasaki H. Ahmed S.A. Morita H. Morita H. Nagata S. J. Biol. Chem. 1989; 264: 6280-6287Abstract Full Text PDF PubMed Google Scholar) and E109A (36Kayastha A.M. Sawa Y. Nagata S. Kanzaki H. Miles E.W. Indian J. Biochem. Biophys. 1991; 28: 352-357PubMed Google Scholar) forms of the tryptophan synthase α2β2 complex and wild type β2 subunits (37Yang X.-J. Ruvinov S.B. Miles E.M. Protein Expr. Purif. 1992; 3: 347-354Crossref PubMed Scopus (17) Google Scholar) from Salmonella typhimurium were isolated and purified to homogeneity. Protein concentrations were determined from the specific absorbance at 278 nm of the α2β2 complex (E1% = 6.0) or of the β2 subunit (E1% = 6.5)(38Miles E.W. Bauerle R. Ahmed S.A. Methods Enzymol. 1987; 142: 398-414Crossref PubMed Scopus (95) Google Scholar). One unit of activity in any reaction is the formation of 0.1 μmol of product in 20 min at 37°C. β-Replacement reactions with L-serine or β-chloro-L-alanine and indole were measured by a direct spectrophotometric assay (38Miles E.W. Bauerle R. Ahmed S.A. Methods Enzymol. 1987; 142: 398-414Crossref PubMed Scopus (95) Google Scholar) containing modified components (100 mML-serine or 40 mM β-chloro-L-alanine, respectively, and 0.2 mM indole in Buffer B). Absorption spectra were made using a Hewlett-Packard 8452 diode array spectrophotometer. Time course measurements of enzymatic activities at single wavelengths were made using a Cary 118 spectrophotometer. The results reported here were all carried out in 50 mM sodium N,N-bis(2-hydroxyethyl)glycine containing 1 mM EDTA at pH 7.8, since we had used this buffer for previous related experiments(17Ahmed S.A. Ruvinov S.B. Kayastha A.M. Miles E.W. J. Biol. Chem. 1991; 266: 21548-21557Abstract Full Text PDF PubMed Google Scholar). Although Na+ does not activate the separate β2 subunit(22Crawford I.P. Ito J. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 390-397Crossref PubMed Scopus (47) Google Scholar, 23Hatanaka M. White E.A. Horibata K. Crawford I.P. Arch. Biochem. Biophys. 1962; 97: 596-606Crossref PubMed Scopus (39) Google Scholar), recent studies show that Na+ does activate the wild type α2β2 complex(27Dunn M.F. Brzovic' P.S. Leja C. Pan P. Woehl E.U. Marino G. Sannia G. Bossa F. Biochemistry of Vitamin B and PQQ. Birkhauser Verlag, Basel/Switzerland1994: 119-124Google Scholar, 28Peracchi A. Mozzarelli A. Rossi G.L. Marino G. Sannia G. Bossa F. Biochemistry of Vitamin B and PQQ. Birkhauser Verlag, Basel/Switzerland1994: 125-129Google Scholar, 29Peracchi A. Mozzarelli A. Rossi G.L. Biochemistry. 1995; (in press)PubMed Google Scholar, 30Woehl E.U. Dunn M.F. Coordination Chemistry Reviews. 1995; (in press)Google Scholar, 31Woehl E.U. Dunn M.F. Biochemistry. 1995; (in press)Google Scholar). These recent studies utilized triethanolamine and bis-tris propane buffers that contain no monovalent cations and are thus better suited for studies of the effects of cations. Because Na+ in our buffer (∼50 mM) may compete with other cations for the same binding site, our results do not show the effects of the absolute concentrations of other cations. However, our results do show that other cations have striking effects on the properties of the wild type β2 subunit and α2β2 complex and on the E109A α2β2 complex. Table 2 shows the effects of various monovalent cations as their chlorides (0.2 M) on the specific activity of the wild type β2 subunit in the β-replacement reactions with L-serine and indole and with β-chloro-L-alanine and indole. Addition of Li+ or NH4+ significantly stimulates activity with L-serine and indole, as found previously with partially purified β2 subunit from Escherichia coli(23Hatanaka M. White E.A. Horibata K. Crawford I.P. Arch. Biochem. Biophys. 1962; 97: 596-606Crossref PubMed Scopus (39) Google Scholar). Stimulation of the β2 subunit by Rb+ and Cs+ is reported for the first time. Because Cs+ gave the greatest stimulation, Cs+ was selected for further study. The monovalent cations have much smaller effects on the activities with β-chloro-L-alanine plus indole. Thus the monovalent cations that increase the activity with L-serine and indole alter the substrate specificity by increasing the ratio of activity with L-serine to activity with β-chloro-L-alanine (see serine:β-chloro-L-alanine activity ratio in Table 2). Note that the ratio of activities with L-serine and β-chloro-L-alanine is one of the features that distinguish the postulated conformations I and II (Table 1).TABLE II Open table in a new tab Fig. 1 shows the effects of CsCl concentration on activity in the β-replacement reaction with L-serine and indole. Low concentrations of CsCl activate the wild type α2β2 complex (A) and β2 subunit (B). High concentrations of CsCl activate the E109A α2β2 complex (C) but inhibit the wild type α2β2 complex (A). The α subunit ligand, GP, alters the effect of CsCl concentration on the activities of the wild type and E109A α2β2 complexes. The activity data in Fig. 1 can be represented by a model (see Table 3) which assumes the noncooperative binding of CsCl to one or two classes of sites on the protein with average dissociation constants K1 and K2. The binding of CsCl converts the enzyme from the CsCl free form X to CsCl-bound forms Y or Z, with concomitant changes in activity. Addition of higher concentrations of CsCl (100-3000 mM) to the wild type α2β2 complex (Fig. 1A) produces further changes of the activity which vary monotonically with salt concentration. These changes could result from additional binding at very weak sites on the protein or from the non-ideal behavior of concentrated salt solutions. For the purpose of curve fitting, these changes are expressed as a linear function of salt concentration and described as a "solvent effect" in Table 3. The curves shown in Fig. 1, A-C, were the best fits to this model. Table 3 describes the model and the derived dissociation constants. The results give no indication of the stoichiometry of the reaction between CsCl and the enzyme.TABLE III Open table in a new tab The absorption spectra of the wild type α2β2 complex (Fig. 2A), β2 subunit (Fig. 2B), and E109A α2β2 complex (Fig. 2C) are very similar in the absence of L-serine. Each enzyme exhibits a major peak centered at 412 nm due to the internal aldimine formed between pyridoxal phosphate and β2 subunit Lys-87 (E in Fig. SI). Reaction of the wild type α2β2 complex with L-serine in the presence or absence of CsCl yields a complex spectrum with a major peak centered at 340 nm (Fig. 2A), which is ascribed to the pyridoxal phosphate-amino acrylate Schiff base (E-AA in Fig. SI). Reaction of the β2 subunit (Fig. 2B) or E109A α2β2 complex (Fig. 2C) with L-serine in the absence of CsCl yields an absorption spectrum with maximum absorbance at 424 nm. This intermediate is the external aldimine of pyridoxal phosphate with L-serine (E-Ser in Fig. SI) and exhibits an intense fluorescence emission at 510 nm. Addition of increasing concentrations of CsCl to the β2 subunit (Fig. 2B) or the E109A α2β2 complex (Fig. 2C) results in decreased absorbance at 424 nm, increased absorbance at 340 nm, and decreased fluorescence emission at 510 nm (fluorescence data not shown). Thus CsCl alters the equilibrium distribution of ES-Ser and ES-AA intermediates with the β2 subunit and the E109A α2β2 complex.Figure SI:Scheme I. Reactions of the wild type tryptophan synthase α2β2 complex.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Globular proteins are flexible molecules and can have two or more conformations in solution. Ligand or substrate binding may produce changes in protein conformation or alter the equilibrium distribution of preexisting conformations. The results reported above show the effects of two ligands (Cs+ and α-glycerol 3-phosphate) on the catalytic properties and spectroscopic properties of wild type and mutant forms of tryptophan synthase. We conclude that Cs+ stabilizes the active conformation of tryptophan synthase and partially repairs the deleterious effects of a mutation that leads to an inactive conformation. Our analysis (Table 3) of the activity data in Fig. 1 indicates that Cs+ binds to two classes of sites in the wild type and E109A α2β2 complexes and to one class of sites in the wild type β2 subunit. Low concentrations of Cs+ activate the wild type α2β2 complex (Kd = 25 mM) and the wild type β2 subunit (Kd = 43 mM). Because ∼50 mM Na+ is present in our assay mixture, the activity of the wild type α2β2 complex in the absence of CsCl is significantly higher than the activity in the absence of any monovalent cation(27Dunn M.F. Brzovic' P.S. Leja C. Pan P. Woehl E.U. Marino G. Sannia G. Bossa F. Biochemistry of Vitamin B and PQQ. Birkhauser Verlag, Basel/Switzerland1994: 119-124Google Scholar, 28Peracchi A. Mozzarelli A. Rossi G.L. Marino G. Sannia G. Bossa F. Biochemistry of Vitamin B and PQQ. Birkhauser Verlag, Basel/Switzerland1994: 125-129Google Scholar, 29Peracchi A. Mozzarelli A. Rossi G.L. Biochemistry. 1995; (in press)PubMed Google Scholar, 30Woehl E.U. Dunn M.F. Coordination Chemistry Reviews. 1995; (in press)Google Scholar, 31Woehl E.U. Dunn M.F. Biochemistry. 1995; (in press)Google Scholar). The cited results indicate that monovalent cations convert the wild type α2β2 complex from conformation I that is less active in the β reaction to a more active conformation II as shown in Table 1. Our results in Table 3 indicate that low concentrations of Cs+ convert the wild type α2β2 complex from a less active form X to a more active form Y. High concentrations of Cs+ decrease the activity of the wild type α2β2 complex. The shape of the curve in Fig. 1A (-GP) suggests that these high concentrations of CsCl may have a solvent effect and favor a less active conformation Z (Table 3). Since 3 M CsCl does not alter the far- and near-UV circular dichroism spectra of the wild type α2β2 complex (data not shown), effects of this high concentration of CsCl must have subtle, rather than gross, effects on the conformation. Addition of GP, an analogue of the α subunit substrates indole-3-glycerol phosphate and D-glyceraldehyde 3-phosphate, inhibits the activity of the wild type α2β2 complex in the β reaction (Fig. 1A) as found previously(39Miles E.W. J. Biol. Chem. 1991; 266: 10715-10718Abstract Full Text PDF PubMed Google Scholar). Inhibition is greatest near the concentration of CsCl that gives maximum activity in the absence of GP. This result suggests that the most active form of the α2β2 complex (form Y in Table 3) is most susceptible to this allosteric inhibition. It is possible that the step which is inhibited by α-glycerol 3-phosphate is rate-limiting for form Y of the α2β2 complex but is only partially rate-limiting for forms X and Z. Our finding CsCl alters the equilibrium distribution of enzyme-substrate intermediates (ES-Ser and ES-AA in Fig. SI) with the β2 subunit (Fig. 1B) is consistent with early studies on the effects of NH4+ on the spectroscopic properties of the β2 subunit with L-serine(24Goldberg M.E. York S. Stryer L. Biochemistry. 1968; 7: 3662-3667Crossref PubMed Scopus (76) Google Scholar). NH4+ was also found to alter the presteady state kinetics of interconversion of ES-Ser and ES-AA(26York S.S. Biochemistry. 1972; 11: 2733-2740Crossref PubMed Scopus (42) Google Scholar). In some ways high concentrations of NH4+ mimic the effects of the α subunit on the properties of the β2 subunit. NH4+ or α subunit increases the rate of formation of E-Ser and increases the rate of conversion of E-Ser to E-AA (Fig. SI). The conversion of E-Ser to E-AA is rate-limiting in the absence of NH4+ or α subunit(25Miles E.W. McPhie P. Biochemistry. 1974; 249: 2852-2857Google Scholar). Addition of Cs+ to the α2β2 complex having the E109A mutation in the β2 subunit partially restores the activity with L-serine and indole (Fig. 1C) and makes the spectroscopic properties in the presence of L-serine (Fig. 2C) more like those of the wild type α2β2 complex (Fig. 2A). These results provide evidence that a high concentration of Cs+ stabilizes conformation II of the E109A α2β2 complex that is more active in the β reaction. Alternatively, high concentrations of Cs+ might prevent dissociation of the E109A α2β2 complex to separate α and β2 subunits under assay conditions. A strong argument against this possibility is provided by our previous finding that addition of GP results in an 80% inhibition of the activity of the wild type and E109A α2β2 complexes in the β-replacement reaction with β-chloro-L-alanine and indole, but does not inhibit the corresponding activity of the separate β2 subunit(17Ahmed S.A. Ruvinov S.B. Kayastha A.M. Miles E.W. J. Biol. Chem. 1991; 266: 21548-21557Abstract Full Text PDF PubMed Google Scholar). Related previous studies have shown that addition of a high concentration of NH4+ partially restores the activity of a mutationally altered form of the β2 subunit found in E. coli strain A2B17(22Crawford I.P. Ito J. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 390-397Crossref PubMed Scopus (47) Google Scholar). This type of mutant has been termed "repairable" because it has activity with L-serine in the β reaction in the presence, but not in the absence, of the α subunit. Ammonium ions often work synergistically with α subunits in the activation of other repairable β2 subunits(40Crawford I.P. Johnson L.M. Genetics. 1963; 48: 725-736Crossref PubMed Google Scholar). A high concentration of NH4+ also partially restores the activity of a mutant form of the β2 subunit (B8) that has an amino acid substitution in the "hinge" region(41Zhao G.-P. Somerville R.L. J. Biol. Chem. 1992; 267: 526-541Abstract Full Text PDF PubMed Google Scholar, 42Zhao G.-P. Somerville R.L. J. Biol. Chem. 1993; 268: 14921-14931Abstract Full Text PDF PubMed Google Scholar). The results suggest a functional role for the hinge region in the process of conformational switching. Studies in progress in our laboratory reveal that moderate concentrations of Cs+ or NH4+ also partially restore the activities of a number of other mutant forms of the β2 subunit and α2β2 complex. Taken together the results of these studies support the model in Table 1 that monovalent cations and the α subunit stabilize conformation II of the wild type and mutant β2 subunits that is more active in the β reaction. It is important to obtain additional evidence for the nature of the conformational change postulated in Table 1. Three other pyridoxal phosphate-dependent enzymes which are activated by monovalent cations (tyrosine phenol-lyase(43Demidkina T.V. Myagkikh I.V. Biochimie (Paris). 1989; 71: 565-571Crossref PubMed Scopus (20) Google Scholar, 44Toraya T. Nihira T. Fukui S. Eur. J. Biochem. 1976; 69: 411-419Crossref Scopus (25) Google Scholar), tryptophanase(44Toraya T. Nihira T. Fukui S. Eur. J. Biochem. 1976; 69: 411-419Crossref Scopus (25) Google Scholar, 45Morino Y. Snell E.E. J. Biol. Chem. 1967; 242: 2800-2809Abstract Full Text PDF PubMed Google Scholar, 46Suelter C.H. Snell E.E. J. Biol. Chem. 1977; 252: 1852-1857Abstract Full Text PDF PubMed Google Scholar), and dialkylglycine decarboxylase(47Aaslestad H.G. Bouis Jr., P.J. Philips A.T. Larson A.D. Snell E.E. Braunstein A.E. Severin E.S. Torchinsky Y.M. Pyridoxal Catalysis: Enzymes and Model Systems. Wiley-Interscience, New York1968: 479-490Google Scholar)) have recently been examined by x-ray crystallography(48Isupov M.N. Antson A.A. Dodson G.G. Dementieva I.S. Zakomirdina L.N. Harutyunyan E.H. Marino G. Sannia G. Bossa F. Biochemistry of Vitamin B and PQQ. Birkhauser Verlag, Basel/Switzerland1994: 183-185Google Scholar, 49Toney M.D. Hohenester E. Keller J.W. Jansonius J.N. J. Mol. Biol. 1995; 245: 151-179Crossref PubMed Scopus (103) Google Scholar, 50Antson A.A. Demidkina T.V. Gollnick P. Dauter Z. Von Tersch R.L. Long J. Berezhnoy S.N. Phillips R.S. Harutyunyan E.H. Wilson K.S. Biochemistry. 1993; 32: 4195-4206Crossref PubMed Scopus (130) Google Scholar, 51Toney M.D. Hohenester E. Cowan S.W. Jansonius J.N. Science. 1993; 261: 756-759Crossref PubMed Scopus (177) Google Scholar, 52Hohenester E. Keller J.W. Jansonius J.N. Biochemistry. 1994; 33: 13561-13570Crossref PubMed Scopus (58) Google Scholar). (For an excellent recent review, see (30Woehl E.U. Dunn M.F. Coordination Chemistry Reviews. 1995; (in press)Google Scholar)). Because the available x-ray structures show that the monovalent cation binding sites are not located in the active site, the bound cations should be classified as allosteric effectors(30Woehl E.U. Dunn M.F. Coordination Chemistry Reviews. 1995; (in press)Google Scholar). These allosteric effectors may activate the enzymes by stabilizing the active conformation or may play a more dynamic role. For an enzyme such as tryptophan synthase that catalyzes a reaction through a series of steps (see Fig. SI) monovalent cation binding could selectively lower the activation energy for a particular step and thus alter the equilibrium distribution of intermediates and the kinetics of specific steps(30Woehl E.U. Dunn M.F. Coordination Chemistry Reviews. 1995; (in press)Google Scholar). The crystal structure of pyridoxal phosphate-dependent 2,2-dialkylglycine decarboxylase reveals the location of two binding sites for alkali metal ions. One is located near the active site and accounts for the dependence of activity on K+ or Rb+(49Toney M.D. Hohenester E. Keller J.W. Jansonius J.N. J. Mol. Biol. 1995; 245: 151-179Crossref PubMed Scopus (103) Google Scholar, 51Toney M.D. Hohenester E. Cowan S.W. Jansonius J.N. Science. 1993; 261: 756-759Crossref PubMed Scopus (177) Google Scholar, 52Hohenester E. Keller J.W. Jansonius J.N. Biochemistry. 1994; 33: 13561-13570Crossref PubMed Scopus (58) Google Scholar, 53Hyde C.C. Ahmed S.A. Padlan E.A. Miles E.W. Davies D.R. J. Biol. Chem. 1988; 263: 17857-17871Abstract Full Text PDF PubMed Google Scholar). The exchange of Na+ for K+ at the location near the active site results in a gross change in the coordination geometry, in concerted rearrangement of the conformations of Ser50 and Tyr301, and in small structural changes extending far beyond the immediate surroundings of the metal ion(49Toney M.D. Hohenester E. Keller J.W. Jansonius J.N. J. Mol. Biol. 1995; 245: 151-179Crossref PubMed Scopus (103) Google Scholar, 51Toney M.D. Hohenester E. Cowan S.W. Jansonius J.N. Science. 1993; 261: 756-759Crossref PubMed Scopus (177) Google Scholar, 52Hohenester E. Keller J.W. Jansonius J.N. Biochemistry. 1994; 33: 13561-13570Crossref PubMed Scopus (58) Google Scholar). The changes in the conformations of Ser50 and Tyr301, which are in the active site, may disrupt the productive binding of substrate. This change in conformation could drive the enzyme into an inactive (or low activity form) in which the bound substrate and the active site functional groups and/or the pyridoxal phosphate ring are improperly aligned for catalysis(30Woehl E.U. Dunn M.F. Coordination Chemistry Reviews. 1995; (in press)Google Scholar). These important studies provide the groundwork for understanding the structural and functional roles of monvalent cations in other pyridoxal phosphate enzymes. The three-dimensional structure of the tryptophan synthase α2β2 complex from S. typhimurium has been reported (53Hyde C.C. Ahmed S.A. Padlan E.A. Miles E.W. Davies D.R. J. Biol. Chem. 1988; 263: 17857-17871Abstract Full Text PDF PubMed Google Scholar) and described in several reviews(3Miles E.W. Adv. Enzymol. Relat. Areas Mol. Biol. 1991; 64: 93-172PubMed Google Scholar, 5Miles E.W. Biswas B.B. Roy S. Subcellular Biochemistry; Proteins: Structure, Function, and Protein Engineering. Plenum Press, New York1995: 207-254Google Scholar, 30Woehl E.U. Dunn M.F. Coordination Chemistry Reviews. 1995; (in press)Google Scholar). Because the active sites of the α and β2 subunits are ∼25 Å apart, the observed effects of ligands that bind to each subunit on the properties of the partner subunit are allosteric in nature. Thus ligands that bind to the active site of the α subunit produce allosteric effects on the individual steps in reactions catalyzed by the β2 subunit (Fig. SI). Studies by Peracchi et al.(28Peracchi A. Mozzarelli A. Rossi G.L. Marino G. Sannia G. Bossa F. Biochemistry of Vitamin B and PQQ. Birkhauser Verlag, Basel/Switzerland1994: 125-129Google Scholar, 29Peracchi A. Mozzarelli A. Rossi G.L. Biochemistry. 1995; (in press)PubMed Google Scholar) and Dunn (27Dunn M.F. Brzovic' P.S. Leja C. Pan P. Woehl E.U. Marino G. Sannia G. Bossa F. Biochemistry of Vitamin B and PQQ. Birkhauser Verlag, Basel/Switzerland1994: 119-124Google Scholar, 30Woehl E.U. Dunn M.F. Coordination Chemistry Reviews. 1995; (in press)Google Scholar, 31Woehl E.U. Dunn M.F. Biochemistry. 1995; (in press)Google Scholar) and our studies reported herein show that monovalent cations also act as allosteric effectors of these β2 subunit reactions. In addition, Woehl and Dunn (30Woehl E.U. Dunn M.F. Coordination Chemistry Reviews. 1995; (in press)Google Scholar, 31Woehl E.U. Dunn M.F. Biochemistry. 1995; (in press)Google Scholar) find that monovalent cations are essential for the allosteric activation of the α site by formation of E-AA (see Fig. SI) at the β site. The cited results and results herein support a role for monovalent cations as a switch that converts the low activity conformation I to a high activity conformation II (Table 1). Crystallographic studies of the tryptophan synthase α2β2 complex in progress show that Na+, K+, or Cs+ binds to a site near, but not in, the active site of the β2 subunit.2 2D. R. Davies, personal communication. The results of these studies should give new insights into the roles of monovalent cations in tryptophan synthase. In conclusion, to explain our finding that the wild type β2 subunit of tryptophan synthase and a mutant form of the α2β2 complex (β2 subunit-E109A) have no or low activity in reactions with L-serine but high activity in reactions with β-chloro-L-alanine, we propose that that these enzymes exist in a conformation (I) that results in the improper alignment of the weak hydroxyl leaving group of L-serine for β-elimination. We suggest that bound Cs+ stabilizes conformation II in which the hydroxyl group of L-serine is properly aligned for β-elimination. Thus Cs+ may stabilize alternative conformations of the wild type β2 subunit and E109A α2β2 complex that have structural and catalytic properties more similar to those of the wild type α2β2 complex.
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